DNA encoding epithelins

ABSTRACT

A novel family of growth regulatory proteins termed &#34;epithelins&#34; are described. The epithelins comprise several distinct members sharing significant structural homology. Two members of the epithelin family, epithelin 1 and epithelin 2, have been purified from natural sources. In addition, cDNA and PCR clones encoding mature and precursor epithelins from various chordate sources have been obtained and sequenced, including the complete human, mouse and rat epithelin precursors. The recombinant expression of rat epithelin precursor and mature forms is described. Purified epithelin 1 is a bifunctional growth regulator, capable of stimulating the growth of some cell types while inhibiting the growth of others. Purified epithelin 2 is functionally similar to epithelin 1 with respect to growth inhibitory bioactivity. In contrast, however, epithelin 2 is apparently not capable of eliciting the growth stimulatory activity characteristic of epithelin 1 and, in fact, antagonizes this epithelin 1 activity.

This application is a divisional of application Ser. No. 07/668,648,filed Mar. 13, 1991, U.S. Pat. No. 5,416,192, which is a continuation inpart of application Ser. No. 07/504,508, filed Apr. 3, 1990, nowabandoned, the contents of which are hereby incorporated by reference.

1. Introduction

2. Background of the Invention

3. Summary of the Invention

4. Brief Description of the Figures

5. Detailed Description of the Invention

5.1. Production of Epithelins

5.1.1. Isolation and Purification of Epithelins From Natural CellSources

5.1.2. Chemical Synthesis of Epithelins

5.1.3. Synthesis of Epithelins Using Recombinant DNA Technology

5.1.3.1. Isolation or Generation of Epithelin Genes

5.1.3.2. Construction of Epithelin Expression Vectors

5.1.3.3. Identification of Transfectants or Transformants ExpressingEpithelin Gene Products

5.1.4. Epithelin Derivatives, Analogs and Peptides

5.2. Anti-Epithelin Antibodies

5.3. Biological Profile of the Epithelins

5.4. Uses of the Epithelins, Epithelin-Encoding Nucleic Acid Molecules,Anti-Epithelin Antibodies and Epithelin Receptors

5.4.1. Epithelin Proteins

5.4.2. Epithelin-Encoding Nucleic Acid Molecules

6. Example: Preparation of Purified Epithelin 1 and And Epithelin 2 FromRat Kidney

6.1. Purification Procedures

6.1.1. Acid Ethanol Extraction

6.1.2. Preparative Gel Permeation Chromatography

6.1.3. Reversed-Phase HPLC of Preparative TSK-250 Fractions

6.1.4. Further Purification of Epithelin 1 By Reversed-Phase and GelPermeation HPLC

6.1.5. Further Purification of Epithelin 2 By Reversed-Phase and GelPermeation HPLC

6.2. Bioassays

6.2.1. Cell Growth Inhibitory Assay Using ¹²⁵ I-deoxyuridineIncorporation Into DNA

6.2.2. Cell Growth Inhibitory and Stimulatory Assay Using MurineKeratinocytes

6.2.3. Soft Agar Colony Assay

6.3. Primary Structure Determinations

6.3.1. Reduction and S-Pyridylethylation

6.3.2. Enzymatic Cleavage of SPE-Epithelin 1 and SPE-Epithelin 2

6.3.3. Peptide Isolation

6.3.4. Amino Acid Analysis

6.3.5. Amino Acid Sequence Determination

6.3.6. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

6.4. Characteristics of the Epithelins I and II

6.4.1. Physical and Chemical Properties

6.4.2. Purification of Epithelin 1 and Epithelin 2 and Certain PhysicalProperties

6.4.3. Chemical Structure of Epithelin 1 and Epithelin 2

6.4.4. Biological Properties of Epithelin 1 and Epithelin 2

7. Example: cDNA Cloning of the Epithelin Precursor and TransientExpression of Precursor and Mature Forms

7.1 PCR cDNA Cloning

7.2 Expression in COS Cells

1. INTRODUCTION

The present invention relates to a novel family of growth regulatoryproteins which applicants have termed "epithelins", to methods for theproduction of epithelins, and to their diagnostic and therapeutic uses.Applicants have purified two members of the epithelin family, epithelin1 and epithelin 2, from natural cell sources and have isolated cDNAsencoding several different epithelins. Epithelin 1 and epithelin 2 sharesubstantial structural similarity yet are functionally distinctproteins. Epithelin 1 is a bifunctional growth regulator, capable ofstimulating the growth of some cell types while inhibiting the growth ofothers. Epithelin 2 is functionally similar to epithelin 1 with respectto growth inhibitory bioactivity. In contrast, however, epithelin 2 isapparently not capable of eliciting the growth stimulatory activitycharacteristic of epithelin 1 and, in fact, antagonizes this epithelin 1activity.

2. BACKGROUND OF THE INVENTION

Cellular growth and differentiation appear to be initiated, promoted,maintained, and regulated by a multiplicity of stimulatory, inhibitory,and synergistic factors and hormones. The alteration and/or breakdown ofthe cellular homeostasis mechanism seems to be a fundamental cause ofgrowth-related diseases, including neoplasia. Growth modulatory factorsare implicated in a wide variety of pathological and physiologicalprocesses including signal transduction, cell communication, growth anddevelopment, embryogenesis, immune response, hematopoiesis, cellsurvival and differentiation, inflammation, tissue repair andremodeling, atherosclerosis and cancer.

Epidermal growth factor (EGF), transforming growth factor-α (TGFα),platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),nerve growth factor (NGF), transforming growth factor-β (TFGβ), insulingrowth factor I and II (IGF I, IGF II), hematopoietic growth factorssuch as erythropoietin, colony-stimulating factors (CSF 1 and 2),interleukins (IL-1 to 8), interferons (IFN α, β, γ), tumor necrosisfactor α and β (TNF α and β), leukoregulin, oncostatin M, amphiregulin(AR) and other less defined factors are growth and differentiationmodulatory proteins produced by a variety of cell types either undernormal physiological conditions or in response to exogenous stimuli.Most of these factors appear to act in autocrine and paracrine fashions.(For reviews see: Goustin et al., 1986, Cancer Res. 46:1015-1029;Rozengurt, 1986, Science 234:161-166; Pardee, 1987, Cancer Res.47:1488-1491; Sachs, 1986, Sci. Amer. 254:40-47; Marshall, 1987, Cell50:5-6; Melcher and Anderson, 1987, Cell 30:715-720; Namen et al., 1988,J. Exp. Med. 167:988-1002; Baggiolini et al., 1989, J. Clin. Invest.84:1045-1049; Clemens and McNurlan, 1985, Biochem, J. 226:345-360;Nathan, 1987, J. Clin. Invest. 79:319-326; Sporn and Roberts, 1986, J.Clin. Invest. 78:329-332; Old, 1987, Nature 326:330-331; Beutler andCerami, 1987, New Engl. J. Med. 316:379-385; Weinstein, 1987, J. Cell.Biochem. 33:213-224; Zarling et al., 1987, Proc. Natl. Acad. Sci. USA83:9739-9744; Shoyab et al., 1988, Proc. Natl. Acad. Sci. USA,85:6528-6532; Shoyab et al., 1989, Science 243:1074-1076; Sporn andTodaro, 1985, N. Engl. J. Med. 303:878-880; Sporn and Roberts, 1985,Nature 313:745-747).

There is a great deal of interest in isolating, characterizing, anddefining the functional mechanisms of growth modulatory factors becauseof their potential use in the diagnosis, prognosis, and treatment ofcancer. Moreover, acquiring knowledge of these factors will aid in theunderstanding of the basic mechanisms behind normal growth control andthe loss thereof in cancer cells.

3. SUMMARY OF THE INVENTION

The present invention is directed to epithelins, a novel family of lowmolecular weight, cysteine-rich proteins exhibiting bifunctional growthregulatory activities, to the use of epithelins in the diagnosis andtreatment of human diseases, and to methods for the production ofbiologically active epithelins. Two members of the epithelin family,epithelin 1 and epithelin 2, have been identified and purified toapparent homogeneity, enabling applicants to determine the primarystructures, physical properties and functional characteristics of thesenovel growth modulators. Several other members of the epithelin familyhave been identified by cDNA cloning. Epithelin 1 is a polypeptidecomprising 56 amino acids, while epithelin 2 comprises 57 amino acids.Structurally, epithelin 1 and epithelin 2 share 47% homology at theamino acid level and each contains 12 identically positioned cysteineresidues.

Epithelins may be produced by isolation and purification from naturalsources, by chemical synthesis, or by recombinant DNA technology. In aparticular embodiment of the invention, described more fully by way ofexample herein (Section 6,infra), epithelin 1 and epithelin 2 areisolated from rat kidney tissue and subsequently purified to apparenthomogeneity using a combination of gel permeation and reversed phasehigh performance liquid chromatography (HPLC). As further described inSection 6, infra, applicants have thoroughly characterized the purifiedepithelins I and II with respect to their structural, physical,chemical, and functional characteristics.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Preparative gel permeation HPLC of crude extract.

FIG. 2. Preparative reversed phase HPLC of pooled fractions 25-28 from28 runs of FIG. 1.

FIG. 3. Semi-preparative reversed phase HPLC of pooled fractions 55-59from FIG. 2.

FIG. 4. Analytical reversed phase HPLC of pool 1b from previous run.

FIG. 5. Analytical reversed phase HPLC of pool 2b from FIG. 3.

FIGS. 6(A and B). Analytical gel permeation chromatography of theconcentrated fractions 51(6A) and 52(6B) from FIG. 4.

FIGS. 7(A and B). Analytical gel permeation chromatography of theconcentrated fractions 44(7A) and 45(7B) from FIG. 5.

FIG. 8. Semi-preparative reversed phase HPLC of fractions 50-54 fromFIG. 2.

FIG. 9. Analytical reversed phase HPLC of fractions 18-23 from previousrun.

FIGS. 10(A-D). Analytical gel permeation chromatography of fractionsfrom FIG. 9. Chromatography was performed as described in Section 6.1.,infra. (10A) HPLC of concentrated fraction 36; (10B) HPLC ofconcentrated fraction 37; (10C) HPLC of concentrated fraction 38; (10D)rechromatography of pooled fractions 48 and 49 from A-C, thenconcentrated.

FIGS. 11(A-B). Tricene-SDS-PAGE analysis of epithelin 1 and epithelin 2.An 18% minigel (0.75 mm×10 cm×7 cm) was run at room temperature at aconstant voltage of 90 volts for 4.5 hr in a Bio-Rad mini-protein IIelectrophoresis apparatus. Dried samples were suspended in 10 μl samplebuffer (50 nM Tris pH 6.8, 12% glycerol (w/v), 4% SDS, 4%mercaptoethanol (v/v) and 0.01% serva blue G.) incubated at 95° C. forfive minutes and then applied on the gel. The molecular weight markerswere five polypeptides from the cleavage of the horse heart myoglobin bycyanogen bromide (Sigma Chem. Co.). (11A) epithelin 1; (11B) epithelin2.

FIG. 12. Amino acid sequences and alignment of epithelin 1 (amino acidresidues 282 through 335 of SEQ ID NO:2)and epithelin 2 (amino acidresidues 205 thriufg 261 of SEQ ID NO:2) purified from rat kidney. Thestandard single letter code for amino acids is used: Alanine (A);Arginine (R); Asparagine (N); Aspartic acid (D); Cysteine (C); Glutamine(Q); Glutamic acid (E); Glycine (G); Histidine (H); Isoleucine (I);Leucine (L); Lysine (K); Methionine (M); Phenylalanine (F); Proline (P);Serine (S); Threonine (T); Tryptophan (W); Tyrosine (Y); and Valine (V).The peptide sequences used to design oligonucleotide primers and probesare underlined.

FIG. 13. Hydropathy analysis of epithelin 1 and 2 (Kyte and Doolittle).⁻⁻, epithelin 1; .sup.. . . , epithelin 2.

FIG. 14. Dose response curve of epithelin 1 and epithelin 2 on theinhibition of ¹²⁵ I-deoxyuridine incorporation into DNA of A431 cells., epithelin 1; o, epithelin 2.

FIGS. 15(A-B). (15A) Effect of epithelin 1 and epithelin 2 on thestimulation of 125I-deoxyuridine incorporation into DNA of the murinekeratinocyte cell line Balb/MK,, 2,000-4,000 cells were plated per wellin 96 well plates in low-calcium medium containing 5% dialyzed FBS. ThenGSA assays were performed as described in Section 6.2., infra. ,epithelin 1; o, epithelin 2. (15B) Effect of various concentrations ofepithelin 2 on epithelin 1 (20 ng/ml) elicited incorporation of ¹²⁵I-deoxyuridine into DNA of Balb/MK cells.

FIG. 16(A.B). (16A) Effect of epithelin 1 and epidermal growth factor(EGF) on the growth of Balb/MK cells. Assays were performed as describedin Section 6.2., infra. , epithelin 1; o, EGF (16B) Effect of variousconcentrations of epithelin 2 on the epithelin 1 (20 ng/ml) inducedgrowth of murine keratinocytes.

FIG. 17. Effect of epithelin 1 and 2 on NRK-SA6 cell colony formation insoft agar in the presence of TFGβ (1 ng/ml). The colony formation assayused is described in Section 6.2.3, infra. Solid bars, epithelin 1;stippled bar, epidermal growth factor; open bar, epithelin 2; hatchedbar, 20 ng/ml epithelin 1 plus 500 ng/ml epithelin 2.

FIG. 18. Dot matrix alignment of the 589 amino acid rat epithelinprecursor compared against itself. Each point represents a stretch offive out of ten identical residues.

FIGS. 19(A-B). (19A) Composite secondary structure analysis of ratepithelin precursor. (19B) Hydropathy of rat epithelin precursor.

FIGS. 20(A-D). (20A) Protein sequence comparison between the human, rat,and mouse epithelin precursor deduced from cDNA clones. Sequences aredisplayed using the single-letter code with identical residues denotedwith dot:s. Gaps were introduced for optimal alignment and are shown bya dash. The predicted rat epithelin signal sequence is underlined andthe seven cysteine-rich motifs are boxed. Each sequence represents aconsensus based on cDNA and PCR clones isolated from human, rat, ormouse kidney RNA. Arrows mark the boundaries of a 234 bp exon, a regionabsent in one rat cDNA clones. (20B) Amino acid sequences of rat, mouse,and human epithelins. (20C) Consensus cysteine motif conserved among theepithelins. (20D) Alignment of the C-terminal domain (amino acids254-315) of a tomato (Lycopersicon esculentum) thiol protease withepithelin 1 and 2.

FIGS. 21(A-B). Expression of recombinant epithelin in COS cells. (21A)³⁵ S-cysteine labeled supernatants from COS cells transfected with thefollowing cDM8-based expression constructs: lane 1, crEPN1.6 containingthe complete rat epithelin coding region; lane 2, crEPN1.4, containing arat epithelin cDNA isoform lacking a 234 bp exon; lane 3,mock-transfected control. (21B) ³⁵ S-cysteine labeled supernatants fromCOS cells transfected with the following cDM8 expression constructs:lane 1, cβrEPN1, containing a simian TGF-β1 signal sequence precedingthe coding region of mature rat epithelin 1; lane 2, cβrEPN2, a similarplasmid based on rat epithelin 2.

FIG. 22. Dendrogram representation of a cluster analysis between theepithelin cysteine-rich motifs from rat, mouse, and human sources. Belowis a diagram of the epithelin precursor showing the position of the 7motifs within the precursor. The 28 cysteine-rich motifs were aligned onPCGENE (Intellignetics, Inc. Mountain View, Calif.) using the CLUSTALmultiple alignment program. The pairwise similarity scores weretransformed into a difference matrix which was analyzed using the Ward'smethod of cluster analysis (SPSS/PC+, Chicago, Ill.). This method usessquared Eclidean distances to place branch points.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel family of growth regulatoryproteins termed "epithelins". The epithelins appear to comprise severaldistinct members sharing significant structural homology. Two members ofthe epithelin family, epithelin 1 and epithelin 2, have been purifiedfrom natural sources, and cDNAs encoding these and several other membersof the epithelin family have been isolated from rat, human, bovine,murine and chicken, among other cell sources.

More particularly, the invention is directed to each and every member ofthe epithelin family, epithelin derivatives and analogues,epithelin-encoding nucleic acid molecules (e.g., cDNAs, genomic DNAs,RNAs, anti-sense RNAs, etc.), traditional and recombinant DNA basedmethods for the production of epithelins, recombinant epithelinexpression vectors, and diagnostic and/or therapeutic uses of mature andprecursor epithelins, epithelin-encoding nucleic acid molecules,anti-epithelin antibodies and epithelin receptor(s).

5.1. PRODUCTION OF EPITHELINS

The individual epithelins may be produced by several general approaches,including isolation from natural sources, solid phase peptide synthesis,and recombinant DNA technology.

5.1.1. ISOLATION AND PURIFICATION OF EPITHELINS FROM NATURAL CELLSOURCES

Applicants' DNA cloning efforts have revealed that messenger RNAsencoding the various epithelins are expressed in a number of differentcell types; representing a broad species range. Therefore, applicants;anticipate that the individual members of the epithelin family may beisolated from a wide variety of organs, tissues, and/or other cellsources. The epithelins may be separated from each other and purifiedfrom such cell sources by using various separation and purificationtechniques known in the art, including but not limited tochromatographic techniques (e.g., reversed phase liquid, gel permeation,liquid exchange, ion exchange, size exclusion, and affinitychromatography), centrifugation, electrophoretic procedures,differential solubility, etc.

In a specific embodiment of the invention, described more fully by wayof example in Section 6., infra, two members of the epithelin family(epithelins 1 and 2) are isolated from rat kidney tissue andsubsequently purified to apparent homogeneity using, inter alia, acombination of gel permeation and reversed phase high performance liquidchromatographies (HPLC). Epithelins 1 and 2 purified in this manner aresingle chain polypeptides comprising 56 and 57 amino acid residues,respectively, and share significant structural characteristics.Functionally, epithelin 1 appears to be a true bifunctional growthmodulator, capable of stimulating and inhibiting cell growth. Epithelin2 appears functionally distinct inasmuch as it specifically antagonizesthe cell growth stimulatory activity induced by epithelin 1. Likeepithelin 1, though generally to a lesser degree, epithelin 2 is alsocapable of inhibiting cell growth. The functional, structural, physicaland other properties of epithelin 1 and epithelin 2 have been determinedand are described in Section 6.4., infra. The six-step method ofpreparing purified epithelin 1 and epithelin 2 described in Section 6.,infra, and/or modifications thereof, may also be used to isolate othermembers of the epithelin family.

5.1.2. CHEMICAL SYNTHESIS OF EPITHELINS

The individual members of the epithelin family may be produced usingchemical methods to synthesize the corresponding amino acid sequences inwhole or in part. For example, epithelins may be synthesized by solidphase techniques (Stewart and Young, Solid Phase Peptide Synthesis, 2ndedition, 1984). Purification and/or refolding into biologically activeconformations of epithelins synthesized in this manner may beaccomplished by various techniques known in the art. The amino acidcompositions of the synthesized epithelins may be confirmed by aminoacid analysis.

5.1.3. SYNTHESIS OF EPITHELINS USING RECOMBINANT DNA TECHNOLOGY

Biologically active mature and precursor epithelins may be produced bythe expression of epithelin-encoding DNAs in a recombinant host cellsystem. General techniques for the isolation of gene sequences, theconstruction of vectors capable of directing the synthesis of encodedproteins, and the expression and/or secretion of biologically activerecombinant proteins are well known in the art.

Production of an epithelin using recombinant DNA technology may bedivided into a four-step process for the purposes of description: (1)isolation or generation of the coding sequence (gene) for a precursor ormature form of the epithelin; (2) construction of an expression vectorcapable of directing the synthesis of the desired epithelin; (3)transfection or transformation of appropriate host cells capable ofreplicating and expressing the epithelin gene and/or processing the geneproduct to produce the desired epithelin; and (4) identification andpurification of the desired epithelin product.

The cloning of a rat epithelin precursor, its expression, and theexpression of mature rat epithelin 1 and 2 are described by the examplespresented in Section 7., et seq, infra.

5.1.3.1. ISOLATION OR GENERATION OF EPITHELIN GENES

The nucleotide coding sequences of the various individual epithelins, orfunctional equivalents thereof, may be used to construct recombinantexpression vectors which will direct the expression of the desiredepithelin product. Epithelin-encoding nucleotide sequences may beobtained from a variety of cell sources which, produce epithelin-likeactivities or which express epithelin-encoding mRNA. Applicants haveidentified a number of suitable human and murine tissue sources in thisregard, including but not limited to placenta, colon, kidney, testes,adrenal, breast, ovary, duodenum, thymus, and lung tissues.

Epithelin coding sequences may be obtained by cDNA cloning from RNAisolated and purified from such cell sources or by genomic cloning.Either cDNA or genomic libraries of clones may be prepared usingtechniques well known in the art and may be screened for particularepithelin-encoding DNAs with nucleotide probes designed from the knownamino acid sequence of epithelin 1 or epithelin 2 and/or which aresubstantially complementary to any portion of the epithelin gene. Fulllength clones, i.e., those containing the entire coding region of theprecursor or mature epithelin desired may be selected for constructingexpression vectors.

Alternatively, epithelin-encoding DNAs may be synthesized in whole or inpart by chemical synthesis using techniques standard in the art.

Due to the inherent degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same or a functionallyequivalent amino acid sequence may be used in the practice of themethods of the invention. Such alterations of epithelin nucleotidesequences include deletions, additions or substitutions of differentnucleotides resulting in a sequence that encodes the same or afunctionally equivalent gene product. The gene product may containdeletions, additions or substitutions of amino acid residues within thesequence which result in silent changes thus producing a bioactiveproduct. Such amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the resides involved.For example, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups or nonpolar headgroups having similar hydrophilicity values include the following:leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine;serine, threonine; phenylalanine, tyrosine.

5.1.3.2. CONSTRUCTION OF EPITHELIN EXPRESSION VECTORS

In order to express biologically active, mature or precursor forms ofthe various epithelins, an expression vector/host system should bechosen which provides not only for high levels of transcription andtranslation but for the correct processing of the gene product. This maybe especially important when employing the entire coding sequence of anepithelin precursor in the expression contructs since the mature formsof the epithelins appear to be derived from larger precursors viacellular processing events.

A variety of animal/host expression vector systems (i.e., vectors whichcontain the necessary elements for directing the replication,transcription and translation of epithelin coding sequences in anappropriate host cell) may be utilized equally well by the skilledartisan. These include, but are not limited to, virus expressionvector/mammalian host cell systems (e.g., cytomegalovirus, vacciniavirus, adenovirus, and the like); insect virus expression vector/insectcell systems (e.g., baculovirus); or nonviral promoter expressionsystems derived from the genomes of mammalian cells (e.g., the mousemetallothionine promoter). Appropriate host cells include but are notlimited to mammilian cells. For example, transient expression ofmammalian proteins may be achieved using a COS cell host, while stableexpression may be achieved using a CHO cell host.

The expression elements of these vectors vary in their strength andspecificities. Depending on the host/vector system utilized, any one ofa number of suitable transcription and translation elements may be used.For instance, when cloning in mammalian cell systems, promoters isolatedfrom the genome of mammalian cells, (e.g. mouse metallothioninepromoter) or from viruses that grow in these cells, (e.g. vaccinia virus7.5K promoter or Moloney murine sarcoma virus long terminal repeat) maybe used. Promoters produced by recombinant DNA or synthetic techniquesmay also be used to provide for transcription of the inserted sequences.

Specific initiation signals are also required for sufficient translationof inserted protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where an entireepithelin gene including its own initiation codon and adjacent sequencesare inserted into the appropriate expression vectors, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the coding sequence is inserted, exogenoustranslational control signals, including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in phase with thereading frame of the epithelin coding sequences to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof transcription attenuation sequences, enhancer elements, etc.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining the epithelin gene of interest and appropriatetranscriptional/translational control signals. These methods may includein vitro recombinant DNA techniques, synthetic techniques and in vivorecombinations.

For example, in cases where an adenovirus is used as an expressionvector, an epithelin coding sequence may be ligated to an adenoviristranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome that is viable and capable ofexpressing the epithelin in infected hosts. Similarly, the vaccinia 7.5Kpromoter may be used.

An alternative expression system which could be used to expressepithelins is an insect system. In one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes. The virus grows in Spodoptera frugiperda cells.An epithelin coding sequence may be cloned into non-essential regions(for example the polyhedrin gene) of the virus and placed under controlof an AcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of an epithelin coding sequence will result in inactivation ofthe polyhedrin gene and production of non-occluded recombinant virus(i.e., virus lacking the proteinaceous coat encoded by the polyhedringene). These recombinant viruses are then used to infect Spodopterafrugiperda cells in which the inserted gene is expressed.

Retroviral vectors prepared in amphotropic packaging cell lines permithigh efficiency expression in numerous cell types. This method allowsone to assess cell-type specific processing, regulation or function ofthe inserted protein coding sequence.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the fashion desired. Expression from certain promoters can beelevated in the presence of certain inducers, (e.g. zinc and cadmiumions for metallothionein promoters). Therefore, expression of thegenetically engineered epithelins may be controlled. This is importantif the protein product of the cloned foreign gene is lethal to the hostcell. Furthermore, modifications (e.g. glycosylation) and processing(e.g., cleavage) of protein products are important for the function ofthe protein. Different host cells have characteristic and specificmechanisms for the post-translational processing and modification ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the expressed foreignprotein.

5.1.3.3. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS EXPRESSINGEPITHELIN GENE PRODUCTS

The host cells which contain the recombinant coding sequence and whichexpress the biologically active, mature product may be identified by atleast four general approaches (a) DNA-DNA, DNA-RNA or RNA-antisense RNAhybridiation; (b) the presence or absence of "marker" gene functions;(c) assessing the level of transcription as measured by the expressionof epithelin mRNA transcripts in the host cell; and (d) detection of themature gene product as measured by immunoassay and, ultimately, by itsbiological activities.

In the first approach, the presence of epithelin coding sequencesinserted into expression vectors can be detected by DNA-DNAhybridization using probes comprising nucleotide sequences that arehomologous to epithelin coding sequences.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain "marker" gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if an epithelin coding sequence is inserted within a marker genesequence of the vector, recombinants containing that coding sequence canbe identified by the absence of the marker gene function. Alternatively,a marker gene can be placed in tandem with the epithelin sequence underthe control of the same or different promoter used to control theexpression of the epithelin coding sequence. Expression of the marker inresponse to induction or selection indicates expression of the epithelincoding sequence.

In the third approach, transcriptional activity for an epithelin codingregion can be assessed by hybridization assays. For example,polyadenylated RNA can be isolated and analyzed by Northern blot using aprobe homologous to the appropriate epithelin coding sequence orparticular portions thereof. Alternatively, total nucleic acids of thehost cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of the mature protein product canbe assessed immunologically, for example by Western blots, immunoassayssuch as radioimmunoprecipitation, enzyme-linked immunoassays and thelike. The ultimate test of the success of the expression system,however, involves the detection of the biologically active epithelingene product. Where the host cell secretes the gene product, cell freemedia obtained from cultured transfectant host cells is assayed forepithelin activity. Where the gene product is not secreted, cell lysatesmay be assayed for such activity. In either case, biological assays suchas the growth inhibition and stimulation assays described herein or thelike may be used.

5.1.4. EPITHELIN DERIVATIVES, ANALOGS AND PEPTIDES

The production and use of derivatives, analogues, and peptides relatedto the epithelins are also envisioned and are within the scope of theinvention. Such derivatives, analogues, and peptides which exhibitgrowth modulatory activity may, like the various epithelins, findapplications in the diagnosis, prognosis, and treatment of a widevariety of neoplasias and other growth related diseases. Suchderivatives, analogues, or peptides may have enhanced or diminishedbiological activities in comparison to native epithelins and/or mayexpand or limit epithelin growth inhibitory activity (GIA)-susceptiblecell range and still be within the scope of the invention. Similarly,the production and use of derivatives, analogues, and peptides relatedto epithelins which exhibit enhanced or diminished growth stimulatoryactivity (GSA) and/or which expand or limit the range of cellsresponsive to epithelin GSA may find useful applications including, butnot limited to, the treatment of wounds and burns.

Epithelin-related derivatives, analogues, and peptides of the inventionmay be produced by a variety of means known in the art. Procedures andmanipulations at the genetic and protein levels are within the scope ofthe invention.

At the protein level, numerous chemical modifications could be used toproduce epithelin-like derivatives, analogues, or peptides by techniquesknown in the art, including but not limited to acetylation, formylation,oxidation, specific chemical cleavage by endopeptidases (e.g. cyanogenbromide, trypsin, chymotrypsin, V8 protease, and the like) orexopeptidases, etc.

5.2. ANTI-EPITHELIN ANTIBODIES

Also within the scope of the invention is the production of polyclonaland monoclonal antibodies which recognize epithelins or relatedproteins.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of epithelins. For the production ofantibodies, various host animals can be immunized by injection with anepithelin, or a synthetic epithelin peptide, including but not limitedto rabbits, mice, rats, etc. Various adjuvants may be used to increasethe immunological response, depending on the host species, including butnot limited to Freund's (complete and incomplete), mineral gels (such asaluminum hydroxide), surface active substances (such as lysolecithin),pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

A monoclonal antibody to an epitope of an epithelin can be prepared byusing any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include but are notlimited to the hybridoma technique originally described by Kohler andMilstein (1975, Nature 256, 495-497), and the more recent human B-cellhybridoma technique (Kosbor et al., 1983, Immunology Today 4:72) andEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab' fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragment, andthe two Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent.

Antibodies to epithelins may find use in the qualitative detection ofmature epithelins and their precursor and subcomponent forms, in theaffinity purification of epithelin proteins, and in the elucidation ofepithelin biosynthesis, metabolism and function. Antibodies toepithelins may also be useful as diagnostic and therapeutic agents.

5.3. BIOLOGICAL PROFILE OF THE EPITHELINS

Applicants' initial cDNA cloning efforts indicate that the epithelinfamily of growth modulatory proteins is comprised of severalstructurally similar members. Moreover, it appears thatepithelin-encoding mRNA is expressed in several tissue types over abroad range of chordates. Applicants' initial data regarding thestructure of epithelin genes from these various chordate sourcessuggests that the epithelin gene has been in place and has remainedremarkably constant for at least 250 million years. The variousepithelins appear to be single chain low molecular weight proteins. Noneof the sequences obtained for the epithelins are significantlyhomologous to any previously known protein. Interestingly, several ofthe epithelins contain a region homologous with the active site ofphospholipase A2.

Epithelin 1 and epithelin 2 purified from rat kidney tissue are proteinsof 56 and 57 amino acids, respectively, sharing 47% amino acid sequencehomology and 12 identically positioned cysteine residues. Thus, aboutone-fifth of the amino acids of both epithelins are cysteines. Thepositioning of these 12 cysteine residues suggests that, through theformation of disulfide linkages between them, the pattern directs theformation of a "cysteine cage" at the tertiary structural level, perhapsnot unlike zinc finger structures. Applicants are not aware of any otherprotein having this particular, or a closely related, cysteine pattern.This unique cysteine pattern is observed not only in the primarystructures of purified epithelins 1 and 2, but also in the structuresdeduced from the sequences of several epithelin-encoding cDNAs and PCRclones. It appears that this unique cysteine pattern is therefore adistinguishing characteristic of the epithelin family members, andlikely plays an important functional role. In addition, some 15 otheramino acids are conserved between these two purified epithelins.

A cDNA encoding the complete rat epithelin precursor has been isolatedand its nucleotide and deduced amino acid sequences determined, as shownin SEQ ID NOS: 1 and 2. The rat epithelin precursor comprises apolypeptide of 589 amino acid residues, the amino-terminal 17 residuesof which comprise its signal peptide. It appears that at least sevencomplete and distinct epithelin species are encoded within the ratepithelin precursor gene. The high level of sequence homology betweenthe epithelins encoded by the rat epithelin precursor cDNA can bevisualized by the 7.5-fold internal homology generated upon comparisonof the rat epithelin precursor sequence to itself, as illustrated inFIG. 19. Composite secondary structure and hydropathy analyses of therat epithelin precursor are shown in FIG. 19A and FIG. 19B,respectively. Amino acid sequence alignments between the rat, mouse andhuman epithelin precursors (SEQ ID NOS: 2, 6, and 4, respectively) areshown in FIG. 20A.

Applicants have also isolated the complete human, mouse and ratepithelin precursor DNA sequences (SEQ ID NOS: 1, 5, and 3,respectively). A composite alignment of these epithelin sequences isillustrated in FIG. 20A. In addition, a number of PCR clones encodingvarious epithelins of bovine and avian origin have been obtained and thebovine and avian epithelin precursor structures partially determined(SEQ ID NOS: 7 and 8 (bovine) and SEQ ID NOS: 9 and 10 (chicken)).Analysis of the various sequences indicates that the epithelins share,to somewhat different degrees, a distinct structural characteristicdefined by the highly conserved cysteine motif (consensus) shown in FIG.20C. While this consensus sequence appears to be generally conservedamong the epithelins, a cysteine core motif of "CCx₈ CCx₆ CCx₅ CC" isalmost completely conserved in all epithelin species examined to date,regardless of origin. Exceptions include the first full repeat of therat epithelin precursor (SEQ ID NO: 2) where Cysteine to serinesubstitutions are present and the motif is "SCx₉ CCx₆ SCx₅ CC".Furthermore, a histidine residue at the 25th position of the core motifalso appears to be conserved among the epithelin species. Applicantsbelieve that the core motif is a unique characteristic of all epithelinsand, accordingly, intend that the present invention encompass allproteins having this structural feature. FIG. 20B shows the amino acidsequence of human, rat and mouse mature epithelins 1-7 aligned. Thesequence represented by FIG. 21C is completely conserved betweenepithelins 2-7 in these three species. The epithelin 1 sequences divergefrom this sequence only slightly.

Applicants have examined RNA from a large number of human and murinetissues and cell lines for the presence of epithelin transcripts, theresults of which are sumarized in TABLE I, below.

                  TABLE I                                                         ______________________________________                                        DISTRIBUTION OF EPITHELIN TRANSCRIPT EXPRESSION IN                             VARIOUS HUMAN AND MURINE TISSUES AND CELLS                                   ______________________________________                                        HUMAN                                                                                                  Strong + Weak + Negative (using Rat Probe)           ______________________________________                                          Placenta Ovary Brain                                                          Colon Duodenum Epidermis                                                      Kidney Medulla Thymus Liver                                                   Kidney Cortex Lung Pituitary                                                  Testis Kidney Amnion                                                          Adrenal  Bone Marrow                                                          Breast  Cerebellum                                                            CRL 7386 HBL100                                                               Caki-1 HEPM MCF-7                                                             CRL 1550 CRO 1572 HTB27                                                       Caki-2 HTB132 T47D                                                            HUF 1477 CEMA-1                                                               CCL 137 HSB2                                                                   U937 Breast Ca                                                                HTB131 Wilm's Ca                                                              BT474                                                                         HTB36                                                                      ______________________________________                                          MOUSE                                                                         Strong + Weak + Negative                                                    ______________________________________                                          Fetal Intestine Heart NONE                                                    Placenta Ovary                                                                Kidney Thymus                                                                  Pancreas                                                                     Brain (Cortex) Cerebellum                                                      Lung                                                                          Embryo d15                                                                    Embryo D6                                                                     Liver                                                                         Colon                                                                         Duodenum                                                                      Skeletal Muscle                                                               CCL51                                                                         CCL51 (TPA)                                                                ______________________________________                                    

The biological characteristics of purified epithelin 1 and epithelin 2are described in some detail in Section 6.4, infra. Both proteins arestable after treatment with 1M acetic acid, 1M ammonium hydroxide, 6Murea, 10 mM sodium metaperiodate, and heating at 56° C. for 30 minutes.The bioactivities of both proteins are sensitive to, inter alia,proteolytic enzymes and reducing agents. It is clear that at least somedisulfide linkages in the epithelin structure are essential forbioactivity. It does not appear that oligosaccharides and/or lipidmoieties are essential for the activity of either epithelin 1 or 2.

Epithelin 1 and epithelin 2 exhibit growth inhibitory activity on, interalia, human epidermoid carcinoma cells. Applicants' data suggests,however, that epithelin 1 is a more potent inhibitor of cell growth. Forexample, the calculated specific activity of purified epithelin 1 isnearly ten times that of epithelin 2, and epithelin 1 appears to be some36 times more potent than epithelin 2 in inhibiting the growth of A431cells. Moreover, in at least one cell line tested, a human coloncarcinoma cell line, epithelin 2 was incapable of triggering theinhibitory effect observed with epithelin 1.

Of additional interest is the functional divergence between epithelin 1and epithelin 2 with respect to cell growth stimulatory bioactivity.Applicants have demonstrated that epithelin 1, in addition to its growthinhibitory bioactivity, is a potent stimulator of cell growth on severalcell lines, leading applicants to conclude that epithelin 1 is a truebifunctional growth modulator. In contrast, applicants' data suggeststhat epithelin 2 is incapable of triggering a growth stimulatory effect,at least on the cell lines tested.

Perhaps most interesting of all is applicants' further discovery thatepithelin 2 specifically antagonizes the stimulatory activities exertedby epithelin 1. Although the functional interrelationship among theseand/or other epithelins is not understood at the present time, it ispossible that epithelin 2 may function as a control on the stimulatoryactivity induced by epithelin 1. Applicants speculate that anagonist/antagonist functional relationship among epithelin 1 andepithelin 2, and/or among other members of the epithelin family, maycontrol or otherwise influence the balance between normal andunrestrained cell growth and development. In this regard, cellularhomeostatis may be altered or destroyed by the loss of a criticalfunction provided by an epithelin or epithelins involved in such aninterrelationship resulting, for example, in unrestrained cellproliferation. Through the therapeutic use of epithelins, anti-epithelinantibodies, and/or epithelin receptors, cellular homeostasis may bemodulated and/or restored.

The ability to express individual motifs from the epithelin precursorwill assist in determining whether differential processing releasesother active molecules. In order to select candidate effectors orblockers, the sequences of the cysteine-rich motifs shown in FIG. 20Awere aligned and cluster analyzed. The results are represented by thedendrogram in FIG. 22. In all cases, the least dissimilar motif is foundat the same position within the precursor of the other 2 species. Basedon these findings, it is proposed that the primordial epithelin geneunderwent a seven-fold replication prior to the divergence of rodentsand humans. This analysis also shows that the fifth repeat of theepithelin precursor is most similar to epithelin 1, and is a candidatefor having growth stimulatory activity. In addition, the second repeatis most similar to epithelin 2, and is a candidate antagonist of themitogenic effects of epithelin 1.

The epithelin gene has several intriguing features. The gene'subiquitous expression suggests it plays a role in the maintenance ofnormal epithelial cell growth, in contrast to previously describedmolecules that have a more restricted distribution. The highlyrepetitive and cysteine-rich structure of the epithelin precursordefines a novel and evolutionarily conserved motif. Furthermore, atleast two of these motifs can be proteolytically processed into activegrowth regulators. This configuration is similar to that ofproopiomelanocortin (POMC), a prohormone that: is processed in atissue-specific manner to release a variety of bioactive peptides.(Smith and Funder, 1988, Endocr. Rev. 9:159-79).

The opposing activities of epithelin 1 and 2 on the growth of epithelialcells is reminiscent. of other systems where naturally occurring,structurally related molecules act as antagonists or suppressors of theparent molecule. Examples of this include; IL-1ra, an interleukin-1receptor antagnoist (Hannum et al., 1990, Nature 343:336-40; Eisenberget al., 1990, Nature 343:341-46); Krev-1 (Kitayama et al., 1989, Cell56:77-84), a protein that suppresses ras induced transformation; andinhibin (Ling et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:7217-21), agonadal protein that opposes the biological effects of activin (Ling etal., 1986, Nature 321:779-82). Further studies to identify cellularreceptors for epithelin 1 and 2 are needed to define how these moleculesmediate their opposing signals. The finding that both activities areproducts of the same transcript is provocative. Conceivably, tissue-,spatial-, or temporally-specific processing might provide a unique meansof regulating epithelial homeostasis.

5.4. USES OF THE EPITHELINS, EPITHELIN-ENCODING NUCLEIC ACID MOLECULES,ANTI-EPITHELIN ANTIBODIES AND EPITHELIN RECEPTORS

Applicants envision a wide variety of uses for the compositions of thepresent invention, including diagnostic and/or therapeutic uses of theepithelin proteins, epithelin analogues and derivatives,epithelein-encoding nucleic acid molecules, anti-epithelin antibodiesand epithelin receptors.

5.4.1. EPITHELIN PROTEINS

Epithelin proteins, analogues and derivatives, as well as compositionscontaining them, may be used alone or in combination with each otherand/or with other biologically active growth factors, inhibitors, orimmunomodulatory agents to regulate the growth and/or development ofchordate cells in vivo and in vitro.

Different epithelins and epithelin compositions may be used to achievedifferent therapeutic objectives. In particular, given the observedfunctional diversity between epithelin 1 and epithelin 2, applicantsenvision that these two epithelins may be used for different purposes.However, notwithstanding their functional differences, both epithelin 1and epithelin 2 may be useful as anti-tumor agents since they bothdemonstrate the ability to inhibit the growth of neoplastic cells,although applicants' initial data suggests that epithelin 1 may be amore powerful and/or effective tumor inhibitor. The particularcombination of epithelins and/or other factors used will depend on thetype of target cells involved as well as the particular objective(s)desired.

For in vivo use, the subject compositions may be administered in avariety of ways, including but not limited to, injection, infusion,topically, parenterally, etc. Administration may be in anyphysiologically acceptable carrier, including phosphate buffered saline,saline, sterilized water, etc. Epithelins and related molecules may alsobe encapsulated in liposomes and may be conjugated to antibodies whichrecognize and bind to tumor or cell specific antigens, thereby providinga means for "targeting" the compositions of the invention.

The epithelins may be useful in vivo for inducing terminaldifferentiation in tumor cells. Such cells have diverted from theordinary course of cell differentiation characteristic of normal cellsand are capable of continued proliferation. Normal cells, in contrast,differentiate into cells which are incapable, under most circumstances,of further cell division. Thus, the ability of the epithelins toreactivate normal cellular differentiation in tumors and, ultimately, toarrest continued tumor growth may find valuable use in tumor therapyregimens.

Epithelins and related derivatives, analogues, and peptides thereof maybe used alone or with at least one other anti-proliferative compound,including, for example, an interferon, TFG-β, tumor necrosis factors,etc., in the treatment of neoplastic and other growth related diseases.Carcinomas may also be treated by inducing production of epithelins inthe carcinoma cells.

The compounds of the invention may be used in vitro to inhibit thegrowth of cells or cell lines sensitive to epithelins as distinguishedfrom cells which are not sensitive. In this way, heterogeneous mixturesor cell lines can be freed of undesirable cells, where the undesirablecells are sensitive to epithelin growth inhibitory activity. Forexample, the compounds of the invention may be used in vitro toeliminate malignant cells from marrow for autologous marrow transplants,and to eliminate or inhibit the proliferation of malignant cells inblood prior to reinfusion.

The most effective concentration of epithelins for inhibitingproliferation of a given cell may be determined by adding variousconcentrations of epithelins to the tumor cell of interest andmonitoring the amount of inhibition of cell proliferation. The mosteffective concentration of individual inducers and/or combinations ofinducers may be determined by monitoring the production of epithelins inthe carcinoma cells.

Stimulation of cell growth can be induced by epithelin 1, epithelin1-like molecules, and, perhaps, by other members of the epithelinfamily. A wide range of therapeutic applications based on this epithelinbioactivity are envisioned, including but not. limited to wound healingand tissue remodeling. Moreover, antibodies capable of neutralizing theepithelin 1-inhibiting activity of epithelin 2 may also be useful inpromoting wound healing and tissue remodeling, with or without thecoadministration of epithelin 1 or epithelin 1-like molecules.

The compositions of the present invention may also find use in thetreatment of human skin diseases involving the proliferation of normalcells, such as psoriasis. Although the pathogenesis of psoriasis is notknown, the disease involves rapid epithelial cell proliferation andturnover. The accompanying rapid turnover of keratinocytes alterskeratinization, resulting in thickened epidermis and scalescharacteristic of the disease. Since epithelin 1 stimulates the growthand proliferation of keratinocytes, effective therapy inhibiting thisepithelin 1-induced activity may impede the onset and development of thedisease. Therefore, compositions capable of inhibiting what may beendogenous or abnormally high levels of epithelin 1 in psoriasispatients may be effective in curing the disease. Applicants havedemonstrated that epithelin 2 specifically inhibits theepithelin-I-induced stimulation of keratinocytes. In this regard,epithelin 2-containing compositions may be particularly useful in thetreatment of psoriasis. Similarly, antibodies capable of neutralizingepithelin 1 stimulatory activity may be used to inhibit epithelin 1activity.

Applicants also envision the use of epithelins and epithelin-likemolecules for other therapeutic purposes, including but not limited tothe modulation of angiogenesis, renal generation, bone resorption,immune responses, synaptic and neuronal effector functions, thearachidonic cascade, and gonadal and reproductive functions.

A number of diagnostic uses of epithelins and related molecules areenvisioned. In the practice of the invention, the subject polypeptidesmay be joined to a label, such as a radioisotope, enzyme, fluorescer,chemiluminescer, enzyme fragment, particle, etc. Such compounds may beused to titrate the number of epithelin receptors on a cell.Identification of epithelin receptors is an indication of potentialresponsiveness of the cell to the biological effects of epithelins andrelated molecules. Epithelins, epithelin-related molecules, and/orantibodies thereto may be used in competitive assays for detection ofepithelins in media, particularly in physiological media. A wide varietyof diagnostic assays known in the art may be used.

The presence and levels of epithelins in body fluids and tissues maydirectly or inversely relate to the presence and pervasiveness ofcertain cancers and other growth related diseases. Assays which candetect and/or quantify epithelins may find use in diagnosis andprognosis of growth related diseases.

In addition, malignant cells expressing epithelin receptors may bedetected by using labeled epithelins or epithelin-related molecules in areceptor binding assay, or by the use of antibodies to the epithelinreceptor itself. Cells may be distinguished in accordance with thepresence and density of epithelin receptors, thereby providing a meansfor predicting the susceptibility of such cells to the biologicalactivities of epithelins.

5.4.2. EPITHELIN-ENCODING NUCLEIC ACID MOLECULES

Epithelin-encoding nucleic acid molecules or fragments thereof may beused as probes to detect and quantify mRNAs encoding epithelins. Assayswhich utilize nucleic acid probes to detect sequences comprising all orpart of a known gene sequence are well known in the art. Epithelin mRNAlevels may indicate emerging and/or existing neoplasia as well as theonset and/or progression of other human diseases including but notlimited to psoriasis. Therefore, assays which can detect and quantifyepithelin mRNA may provide a valuable diagnostic tool.

Anti-sense epithelin RNA molecules may be useful therapeutically toinhibit the translation of epithelin-encoding mRNAs where thetherapeutic objective involves a desire to eliminate the presence of agiven epithelin. Epithelin 1 anti-sense RNA, for example, could beuseful as an epithelin 1 antagonizing agent in the treatment of diseasesfor which epithelin 1 is a causative agent. Additionally, epithelinanti-sense RNAs may be useful in elucidating epithelin functionalmechanisms.

Epithelin-encoding nucleic acid molecules may be used for the productionof recombinant epithelin proteins and related molecules, as separatelydiscussed in Section 5.1.3., supra.

6. EXAMPLE: PREPARATION OF PURIFIED EPIHELIN 1 AND EPITHELIN 2 FROM RATKIDNEY 6.1. PURIFICATION PROCEDURES 6.1.1. ACID ETHANOL EXTRACTION

Rat kidneys were obtained from Pel-Freeze (Rogers, Arkansas). Frozen ratkidneys (430 g wet weight) were suspended in 2370 ml of extractionbuffer consisting of 2348 ml ethanol (98%), 19 ml of concentrated HCl,81.5 mg phenylmethyl-sulfonyl fluoride and 2.8 ml of aprotonin (23TIU/ml from bovine lung; Sigma Chemical Co.). The tissue was allowed tothaw at 4° C. for 4-6 hours and the mixture was homogenized in a Waringblender. The mixture was stirred at 4° C. overnight, centrifuged at9,000 rpm in a Sorvall GS-3 rotor for 40 minutes and the supernatantcarefully removed (2,200 ml). Chloroform (2,200 ml) and 220 ml ofacidified water (375 ml water+7.5 ml of concentrated HCl) was added tothe supernatant, the mixture stirred vigorously for approximately onehour, and allowed to stand at room temperature to separate into twophases. The upper aqueous phase was carefully removed. and dialyzedagainst 17 liters of 0.1 M acetic acid at 4° C. in No. 3 Spectroporedialysis tubing (molecular weight. cut off approximately 3,000). Thedialysis buffer was changed three times over a two-day period. Theretenate was lyophilized and the lyophilized material (4.55 g), termed"crude extract", was stored at -20° C. until further use.

6.1.2. PREPARATIVE GEL PERMEATION CHROMATOGRAPHY

A Bio-Sil TSK-250 column (21.5×600 mm) (BioRad) was attached to a highperformance liquid chromatography (HPLC) system (Waters). The crudeextract (25 mg/ml) was dissolved in 50% acetonitrile/water with 0.1%trifluoroacetic acid (TFA). A 3 ml aliquot of the mixture was injectedand elution was performed isocratically with a mobile phase of 50%acetonitrile with 0.1% TFA. The flow rate was 4 ml/min and chart speedwas set at 0.25 cm/min. Six ml fractions were collected. Thechromatography was performed at room temperature. An aliquot from eachfraction was evaporated and assayed in triplicate for growth inhibitoryactivity (GIA) on A431 human epidermoid carcinoma cells as described inSection 6.2.1., infra. (FIG. 1).

The late eluting minor peak (Fractions 25-28) contained the newactivities of interest. Fractions 25-28 from 57 similar runs werepooled, concentrated and lyophilized. The lyophilized material weighed473 mg and had a total of approximately 1.1×10⁵ GIA units.

6.1.3. REVERSED-PHASE HPLC OF PREPARATIVE TSK-250 FRACTIONS

The lyophilized fractions (Section 6.1.2., supra) were dissolved in 240ml of 0.1% TFA in water; the mixture was centrifuged, and thesupernatant was carefully removed. The final volume was about 250 ml.125 ml of this mixture was isocratically injected onto a preparativePartisil 10 ODS-3 column (10 micron, 2.2×25 cm; Whatman) attached to aHPLC system. The flow rate was set at 4 ml/min. Once the sample hadpassed onto the column, the cclumn was washed with 150 ml of 0.1% TFA inwater. The linear gradient was generated between the primary solvent,0.1% TFA in water, and the secondary solvent, acetonitrile containing0.1% TFA. The gradient conditions were: 0 to 45% in 270 minutes and 45to 100% in 45 minutes. 14 ml fractions were collected and aliquots ofeach fraction were assayed for GIA. Four broad peaks of activity wereseen (FIG. 2). A second run was performed as described above. Two earlyeluting peaks, peak a and peak b, contained epithelin 2 and epithelin 1,respectively, and they were further purified and characterized. Thefurther purifications of epithelin 1 and epithelin 2 are describedseparately below.

6.1.4. FURTHER PURIFICATION OF EPITHELIN 1 BY REVERSED-PHASE AND GELPERMEATION HPLC

Fractions 55-59 (FIG. 2) from two runs were pooled and diluted twofoldwith 0.1% TFA in water. The mixture was isocratically injected onto asemi-preparative μ-Bondapak-C18 column (7.8×300 mm, Waters) at a flowrate of 2 ml/min at room temperature. The linear gradient conditionsbetween primary solvent, water with 0.1% TFA, and the secondary solvent,acetonitrile with 0.1% TFA, were 0 to 18% in 1.8 minutes, 18 to 18% in20 minutes, 18 to 34% in 240 minutes, and 34 to 100% in 10 minutes. Theflow rate was 2 ml/min throughout the gradient; 7 ml fractions werecollected. Aliquots were taken and assayed for GIA. Two peaks ofactivity were observed eluting at: acetonitrile concentrations ofapproximately 24% and 25%, respectively (FIG. 3).

Fractions 30-34 were pooled. 45 ml of 0.1% TFA in water was added to thepooled fraction. The mixture was isocratically applied onto aμ-Bondapak-CN column (3.9×300 mm, Waters) at a flow rate of 1 ml/min atroom temperature. The gradient conditions were 0 to 10% in 1 minute, 10to 10% in 19 minutes, 10 to 30% in 200 minutes, and 30 to 100% in 7minutes. The flow rate was 0.5 ml/min and 1.5 ml fractions werecollected. Most of the activity emerged from the column at about 21.5%acetonitrile concentration (FIG. 4).

Fractions 36-43 were pooled and diluted with 0.1% TFA/H₂ O to a finalvolume of 115 ml and chromatographed exactly as described for fractions30-34, above. Most of the activity eluted from the column in two peaks,eluting at approximately 22.5% and 23.5% acetonitrile (FIG. 5).

Fractions 51 and 52 (FIG. 4) were individually concentrated, using aspeed-vac concentrator (Savant), to a volume of about 70 μl, to which anequal volume of acetonitrile containing 0.1% TFA was added. This 140 μlsample was injected onto two Bio-Sil TSK-250 columns (7.5×300 mm each,Bio-Rad) arranged in tandem. The elution was performed isocraticallywith a mobile phase of 50% acetonitrile/H₂ O with 0.1% TFA at roomtemperature. The flow rate was 0.4 ml/min and chart speed was 0.25cm/min; 0.4 ml fractions were collected and aliquots were assayed forGIA. The chromatographic profiles of fractions 51 and 52 are shown inFIG. 6A and FIG. 6B, respectively.

Fractions 44 and 45 (FIG. 5) were individually concentrated to 70 μl andthen subjected to gel permeation chromatography as described above. Thechromatographic profiles are given in FIG. 7.

6.1.5. FURTHER PURIFICATION OF EPITHELIN 2 BY REVERSED-PHASE AND GELPERMEATION HPLC

Fractions 50-54 (FIG. 2) from two runs were pooled and diluted twofoldwith 0.1% TFA/H₂ O. The mixture was applied onto a semi-preparativeμ-Bondapak-C18 column (7.8×30 mm, Waters) at a flow rate of 2 ml/min.The linear gradient conditions between primary solvent, water with 0.1%TFA, and the secondary solvent, acetonitrile with 0.1% TFA, were 0 to18% in 1.8 minutes, 18 to 18% in 20 minutes, 18 to 34% in 240 minutes,and 34 to 100% in 10 minutes. The flow rate was 2 ml/min throughout thegradient; 7 ml fractions were collected. Aliquots were taken and assayedfor GIA. The chromatographic profile is shown in FIG. 8. The major peakof activity eluted at approximately 20.5% acetonitrile concentration.

Fractions 18-23 were pooled and diluted with 0.1% TFA/H₂ O to a finalvolume of 110 ml. The mixture was applied onto a μ-Bondapak-CN column(3.9×300 mm, Waters) at a flow rate of 1 ml/min at room temperature Thegradient conditions were 0 to 10% in 1 minute, 10 to 10% in 19 minutes,10 to 30% in 200 minutes, and 30 to 100% in 7 minutes. The flow rate was0.5 ml/min; 1.5 ml fractions were collected. The activity emerged fromthe column at an acetonitrile concentration of about 18% (FIG. 9).

Fractions 36-38 (FIG. 8) were individually concentrated to approximately70 μl, to which an equal volume of acetonitrile containing 0.1% TFA wasadded. This 140 μl sample was applied onto two Bio-Sil TSK-250 columns(7.5×300 mm each, Bio-Rad) attached in tandem. The elution was performedisocratically with a mobile phase of 50% acetonitrile/H₂ O with 0.1%TFA. The flow rate was 0.4 ml/min; 0.4 ml fractions were collected andaliquots were assayed for GIA. The chromatographic profiles of fractions36, 37 and 38 are shown in FIG. 10A, 10B and 10C, respectively.

Fractions 48 and 49 from FIG. 10A-C were pooled and concentrated toabout 70 μl and then subjected to gel permeation chromatography asdescribed above. The rechromatographic profile is presented in FIG. 10D.

6.2. BIOASSAYS 6.2.1. CELL GROWTH INHIBITORY ACTIVITY USING ¹²⁵I-DEOXYURIDINE INCORPORATION INTO DNA

The cell growth inhibitory activity (GIA) assays were performed inflat-bottom 96 well plates (Falcon 3072). Human epidermoid carcinoma ofvulva cells (A431) were used as test cells for GIA. 3.5×10³ cells in 50μl of test medium (DMEM supplemented with 5% heat inactivated fetalbovine serum (FBS), penicillin/streptomycin (PS) and glutamine) wereplaced in all wells except peripheral wells. The peripheral wellsreceived 50 μl PBS. Three hours later, 50 μl of test sample in testmedium was added to each well, while control wells received only 50 μlof test medium. Three wells were used for each concentration of testsample. Plates were incubated at 37° C. for 2-3 days. Then, 100 μl of asolution of ¹²⁵ I-iodo-2'-¹²⁵ I-deoxyuridine (¹²⁵ I-IUdR, 4 Ci/mg-0.5mCi/ml, 2 μl/ml in test medium) was added to each well and plates wereincubated at 37° C. After 4-6 hours, the medium was aspirated from thewells, which were then washed once with 200 μl PBS. Then, 200 μlmethanol was added to each well, plates were incubated for 10 minutes atroom temperature, and the methanol was removed by aspiration. 200 μl of1M sodium hydroxide was added to each well, the plates were incubatedfor 30 minutes at 37° C. Sodium hydroxide was removed with titertekplugs (Flow Labs). The plugs were transferred into 12×75 mm plastictubes and counted in a gamma counter to quantify ¹²⁵ I-IUDRincorporation.

6.2.2. CELL GROWTH INHIBITORY AND STIMULATORY ACTIVITY ASSAYS USINGMURINE KERATINOCYTES

Balb/MK cells were plated at 1×10⁴ cells per well in 1 ml of low calciummedium (Weissman and Aaronson, 1983, Cell 32:599-606; Carpenter andZendegut, 1985, Anal. Biochem. 153:279-282) in 24-well Costar plates andincubated at 37° C. for 4-6 hours. Then media were removed and replacedwith 1 ml of medium containing various concentrations of the testcompound in triplicate. The control wells received only medium withoutany test material. The plates were incubated at 37° C. for 4 days, thenmedium was removed, wells were rinsed two times with 1 ml ofphosphate-buffered saline, and the cells were detached with trypsin-EDTAand counted.

Balb/MK cells were also used as indicator cells in 96 well plates toassess the growth inhibitory activity (GIA) or growth stimulatoryactivity (GSA) of a test material using ¹²⁵ I-deoxyuridine incorporationinto DNA as described in the previous section.

6.2.3. SOFT AGAR COLONY ASSAY

A 0.38 ml base layer of 0.5% agar (Agar Noble; Difco Laboratories,Detroit, Michigan) in DMEM containing 10% heat inactivated FBS was addedto 24 well Costar tissue culture plates. 0.38 ml of 0.3% agar containingthe same medium-FBS mixture, 6-12×10³ test cells, and the test proteinsat various concentrations were overlaid on the basal layer of agar. Theplates were incubated at 37° C. in a humidified atmosphere of 5% CO₂ inair. Colonies were enumerated unfixed and unstained, and the number ofcolonies was scored between days 7 and 10. Colonies were defined as acluster of at least eight cells.

6.3. PRIMARY STRUCTURE DETERMINATIONS 6.3.1. REDUCTION ANDS-PYRIDYLETHYLATION

Protein (10-20 μg) was dried in a 1.5 ml microfuge polypropylene tube,suspended in 100 μl of 3 M urea in 0.05 M Tris-HCl, pH 7.5. Then, 4 μlof 2-mercaptoethanol was added to the mixture, the contents were mixed,flushed with nitrogen, and incubated at 25° C. After 2.5 hours, 4.5 μlof freshly distilled 4-vinylpyridine was added to the mixture, the tubewas again flushed with nitrogen and incubated for 2 hours at 25° C. Thereaction mixture was acidified to pH 2.0 with 10% TFA.S-pyridylethylated protein was purified by reversed phase HPLC using aPartisil 5 ODS-3 column (4.6×100 mm, Whatman). The concentration ofacetonitrile was increased linearly (1%/min) during 55 minutes at a flowrate of 1 ml/min. The primary solvent was 0.1% TFA/H₂ O.S-pyridylethylated-epithelin 1 (SPE-Epithelin 1) and SPE-Epithelin 2eluted at about 25% and 23% of acetonitrile, respectively, approximately2-3% higher acetonitrile concentrations than the untreated epithelins.

6.3.2. ENZYMATIC CLEAVAGE OF SPE-EPITHELIN 1 AND SPE-EPITHELIN 2

Cleavage with endopeptidase Lys-C and TPCK-trypsin was performed in 60μl of 0.1M Tris-acetic acid buffer, pH 8.0 at 25° C. for 16 hours. Theenzyme/substrate ratio was 1 to 5 (w/w). Endopeptidase-Arg and S. aureusV8 protease digestions were done in 80 μl of 0.05M Tris-HCl, pH 8.0,0.1M ammonium-bicarbonate at 37° C. for 16 hours. The enzyme/substrateratio was again 1 to 5.

6.3.3. PEPTIDE ISOLATION

Peptides were separated on a reversed phase HPLC C18 column (4.6×100 mm,Whatman) attached to a HPLC system (Waters). Acidified sample (pH 2.0)was applied onto a column equilibriated with 0.1% TFA (primary solvent)at a flow rate of 1 min/ml and the column was further washed with about15 ml of 0.1% TFA. Linear gradients were used between the primarysolvent and the secondary solvent (acetonitrile with 0.1% TFA). Thegradient conditions were 0 to 50% in 125 minutes at a flow rate of 0.5ml/min.

6.3.4. AMINO ACID ANALYSIS

Dried samples were hydrolyzed with constant boiling HCl (5.7M, Pierce)containing 1% (v/v) phenol under reduced pressure in a Teflon-sealedglass hydrolysis bulb (Pierce) at 105° C. for 16 hr. The hydrolysateswere dried in a Speed Vac concentrator (Savant Instruments) andderivitized with phenylisothiocyanate for 20 minutes at roomtemperature. Phenylthiocarbamyl amino acid derivatives were analyzed byreversed phase HPLC on a Octadecyl column (4.5×250 mm, IBM). The lineargradient was performed between primary solvent 0.15M sodium acetate pH6.4, 0.05% triethylamine titrated to pH 6.4 with acetic acid and thesecondary solent 60% acetonitrile at a flow rate of 1 ml/min at 38° C.

6.3.5. AMINO ACID SEQUENCE DETERMINATION

Peptide sequences were determined with an Applied Biosystem model 475gas phase sequencer as described (Hewick et al., 1981, J. Biol. Chem.256:7990-7997). Three precycles of Edman degradation were performedprior to sample application for each run. 25% TFA was used to convertthe Triazoline derivatives to phenylthiohydantoin amino acids.Identification of phenylthiohydantoin amino acids was carried out,on-line, on a Model 120A PTH analyzer (Applied Biosystem) as described(Hunkapiller and Hood, 1983, Science 219:650-659).

6.3.6. TRICINE-SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS

Proteins were analyzed on tricine/sodium dodecyl sulfate/polyacrylamideslab gels (normal or mini Bio-Rad system) by the method of Schager andGebhard, 1987, Biochem. 166:368-379. Proteins were detected by silverstaining (Wray et al., 1981, Anal. Biochem. 118:197-203).

6.4. CHARACTERISTICS OF THE EPITHELINS 1 AND 2 6.4.1. PHYSICAL ANDCHEMICAL PROPERTIES

Epithelin 1 and epithelin 2 are resistant to treatment with 1M aceticacid, 1M ammonium hydroxide, 6M urea, 0.01M sodium metaperiodate, toheating at 56° C. for 30 minutes, and to treatment with variousglycosidases or lipases. However, epithelin activity was sensitive toreduction, to reduction and 4-vinylpyridine treatment, and to digestionwith proteinases such as trypsin, endoproteinase Lys-C, andendoproteinase Glu-C (V8). These results suggest that these factors areproteins containing cysteines in disulfide linkage(s) that are essentialfor biological activity. These proteins do not contain oligosaccharidesand/or lipid moieties that are obligatory for biological activities.

6.4.2. PURIFICATION OF EPITHELIN 1 AND EPITHELIN 2 AND CERTAIN PHYSICALPROPERTIES

Summaries of the purification of epithelin 1 and epithelin 2 arepresented in Table I and Table II, respectively. Both factors werepurified to apparent homogeneity by a similar six-step protocol. Theearly step fractions contain a multiple of growth inhibitory activitieson A431 cells. Epithelins 1 and 2 constitute only a very minor fractionof total GIA in early fractions, making it very difficult to quantitatetheir specific activities at early stages of purification. The specificactivity of purified epithelin 1 was about 2.1×10⁴ units/mg protein,whereas purified epithelin 2 had a much lower specific activity of3.8×10³ units/mg protein.

                  TABLE II                                                        ______________________________________                                        Summary of Purification of Epithelin 1 (GIA)                                                Protein  GIA     Specific Activity                                                                       Yield                                  Fraction μg Units.sup.1 Units/mg %                                       ______________________________________                                        Crude     4,550,000                                                                              1,283,100.sup.2                                                                           282     --                                       Prep TSK-250 473,000 112,200.sup.2   237 --                                   Prep ODS (b) 17,600 14,100.sup.    801 100                                    Semi Prep. C18                                                                1b 1,510 2,080.sup.   1,377 14.8                                              2b 3,460 5,460.sup.   1,578 38.7                                              Anal. Cyano                                                                   1b 60 713.sup.  11,889 3.4                                                    2b 73 1,190.sup.  16,301 8.4                                                  Anal. Tsk-250                                                                 1b 41 845.sup.  20,609 6.0                                                    2b 62 1,305.sup.  21,048 9.3                                                ______________________________________                                         .sup.1 One unit of GIA is the amount of factor required to inhibit            .sup.125 Ilabeled deoxyuridine incorporation into A431 cells by 50%.          .sup.2 Other growth inhibitory activities are present in these fractions.     These values include all activities.                                     

                  TABLE III                                                       ______________________________________                                        Summary of Purification of Epithelin 2 (GIA)                                                Protein  GIA     Specific Activity                                                                       Yield                                  Fraction μg Units.sup.1 Units/mg %                                       ______________________________________                                        Crude     4,550,000                                                                              1,283,100.sup.2                                                                         282       --                                       Prep TSK-250 473,000 112,200.sup.2 237 --                                     Prep. ODS (b) 24,500 9,567.sup.  432 100                                      Semi Prep. C18 4,760 1,190.sup.  250 12.4                                     Anal. Cyano 169 460.sup.  2,741   4.8                                         Anal TSK-250 37 141.sup.  3,810   1.5                                       ______________________________________                                         .sup.1 One unit of GIA is the amount of factor required to inhibit            .sup.125 Ilabeled deoxyuridine incorporation into A431 cells by 50%.          .sup.2 Other growth inhibitory activities are present in these fractions.     These values include all activities.                                     

The molecular weight of epithelin 1 and eipthelin 2, as determined bygel permeation chromatography on TSK-250 columns, was ˜4,500 and ˜3,700,respectively (FIGS. 6, 7 and 10). SPE-epithelin 1 or 2 exhibited amolecular weight of ˜13,000 by similar gel permeation chromatography.

FIG. 11 shows an analysis of epithelin 1 and epithelin 2 in an 18%polyacrylamide gel under reducing conditions. Epithelin 1 and epithelin2 migrated in the gel as single bands with median relative molecularweights of about 5,500 and 6,000, respectively. Similar results wereobtained when proteins were electrophoresed under nonreducingconditions. Thus, epithelins are single chain, low molecular weightproteins.

6.4.3. CHEMICAL STRUCTURE OF EPITHELIN 1 AND EPITHELIN 2

The amino acid sequences of epithelin 1 and epithelin 2 were deducedfrom microsequence analysis of S-pyridylethylated proteins, andfragments generated by endoproteinase Lys-C, Staphylococcal aureus V8and TPCK trypsin. The amino acid sequences of epithelin 1 and epithelin2 are presented in FIG. 12.

The protein sequences of epithelin 1 and epithelin 2 were compared withall proteins in the National Biomedical Research Foundation data base(release 22), Genetic Sequence Data Bank (Bolt Beranek and Newman, LosAlamos National Laboratory; release 61) and the European MolecularBiology Laboratory data base (release 20). These computer aided searchesrevealed that both proteins are novel and do not share any significanthomology to any protein in the three data bases.

Epithelin 1 and epithelin 2 are single chain polypeptides of 56 and 57amino acid residues, respectively, having calculated molecular weightsof 6060 and 6094, respectively. Epithelin 1 and epithelin 2 share 47%homology at the amino acid level. Both proteins contain 12 cysteineresidues with four double cysteine residues. Thus about 21% of aminoacid residues in both proteins are cysteines. Moreover, the spacing ofsingle cysteine residues and double cysteine residues in epithelin 1 andepithelin 2 is identical. Although the number or position of intrachaindisulfide bonds in these proteins is not presently known, a similar oridentical pattern is expected. Both proteins also contain about 13%hydroxy amino acids (serine and threonine together). The amino acidalignment of epithelin 1 and epithelin 2 is shown in FIG. 12. Inaddition the conservation of cysteine residues, 15 other residues areconserved between epithelin 1 and epithelin 2.

The hydropathy profiles of epithelins 1 and 2 are presented in FIG. 13.The hydropathy profile of epithelin 1 exhibits some similarity to thatof epithelin 2, although epithelin 2 appears to be more hydrophilic thanepithelin 1.

6.4.4. BIOLOGICAL PROPERTIES OF EPITHELIN 1 AND EPITHELIN 2

The inhibition of ¹²⁵ I-deoxyuridine incorporation into DNA of humanepidermoid carcinoma A431 cells by different concentrations of purifiedepithelin 1 and epithelin 2 is given in FIG. 14. A 50% inhibition of DNAsynthesis was seen at 12.8 ng/well of epithelin 1 and 450ng/well ofepithelin 2. Thus, a 50% inhibition occurred at approximately 21 nMconcentration of epithelin 1 and approximately 0.75 μm concentration ofepithelin 2. Epithelin 1 is therefore about 36 times more potent thanepithelin 2 in this assay.

The effect of epithelin on the incorporation of ¹²⁵ I-deoxyuridine intoDNA of various tumor and non-tumor human cell lines, as well as severalnon-human cell lines, was investigated. Epithelin 1 (20 ng/ml, maximumdose tested) slighly inhibited the growth of human colon carcinoma cellline HCT 116, while epithelin 2 (270 ng/ml, maximum dose tested) did notshow any effect on this cell line. Both proteins significantly inhibitedthe ¹²⁵ I-deoxyuridine incorporation into DNA of mink lung CCL 64 cellsand monkey kidney COS1 cells. Neither protein exhibited any significanteffect on human fibroblasts and several other human tumor cells at themaximum dose tested (20 ng/ml for epithelin 1 and 270 ng/ml forepithelin 2).

The effects of various concentrations of epithelin 1 and epithelin 2 onthe incorporation of ¹²⁵ I-deoxyuridine into DNA of murine keratinocytes(Balb/MK cells) was investigated. Data are presented in FIG. 15A.Epithelin 1 stimulated the ¹²⁵ I-deoxyuridine incorporation in adose-dependent manner, while epithelin 2 did not show any significanteffect. However, epithelin 2 inhibited epithelin 1 elicitedincorporation of ¹²⁵ I-deoxyuridine incorporation to Balb/MK cells (FIG.15B). In this regard, a 50% inhibition was observed at ˜21 nM epithelin2. Thus, epithelin 2 antagonizes the effect of epithelin 1 in thissystem.

The continued growth of a murine keratinocyte cell line, Balb/MK, isdependent on EGF, TGFα or amphiregulin (AR). Balb/MK cells did notproliferate in the absence of EGF or epithelin 1 (FIG. 16A), whereasepithelin 2 did not exhibit any significant effect on the growth ofthese cells. However, epithelin 2 inhibited the epithelin 1-inducedgrowth of Balb/MK cells in a dose-dependent manner (FIG. 16B); a 50%inhibition was observed at ˜7 nM epithelin 2. EGF or TGFA inducesanchorage-independent growth of rat kidney cells NRK-SA6 in the presenceof TFGβ (Roberts et al., 1981, Proc. Natl. Acad. Sci. USA 78:5339-5344).Like EGF, epithelin 1 induced the anchorage-independent growth of NRKcells ir a dose-dependent manner (FIG. 17), while epithelin 2 did not.Epithelin 2 (at a concentration of ˜85 nM) inhibited about 50% of theepithelin 1-induced colony formation in soft agar. Furthermore,epithelin 1, but not epithelin 2, exhibited mitogenic activity on NRKcells in monolayer.

Neither epithelin 1 nor epithelin 2 significantly affected the bindingof ¹²⁵ I-EGF to its receptors, suggesting that the epithelins do notmediate their biological effects through EGF receptors.

7. EXAMPLE: cDNA CLONING OF THE EPITHELIN PRECURSOR AND TRANSIENTEXPRESSION OF PRECURSOR AND MATURE FORMS 7.1. PCR cDNA CLONING

Two pools of degenerate oligonucleotides were synthesized based on thepeptide sequences KTQCPDD and HCCPQDT from epithelin 2 (the poolscontained 256 and 128 degenerate oligonucleotides in the sense andantisense orientation, respectively). These oligonucletides were used asprimers in a 40 cycle PCR amplification with a single stranded cDNAtemplate derived from rat kidney RNA primed with XSCT17, anoligonucleotide containing a T₁₇ track on its 3'-end (Plowman et al,1990, Proc. Natl. Acad. Sci. U.S.A. 87:4905-08). An oligonucleotideprobe, based on the epithelin 2 sequence KYGCCPMP (256 fold degenerate23-mer), was labeled with [γ-³² P]ATP and used to screen the PCRproducts.

Hybridization of this probe to a Southern blot of the PCR productsrevealed two major bands of 120 base pairs (bp) and 600 bp, and a minorband of 325 bp. These fragments were subcloned and sequenced, revealingthat they all had a common 5' end encoding epithelin 2. The 325 bpfragment had an open reading frame that encoded both epithelin 1 and 2,whereas the 600 bp band extended further 3' and contained an additionalcopy of the cysteine-rich motif. Therefore, epithelin 1 and 2 appear tobe tandemly arranged products of a single transcript. These same PCRprimers were used to isolate similar fragments of the epithelinprecursor from human, bovine, mouse, and chicken cDNA, demonstratingstrong evolutionary conservation of the cysteine-rich motif.

The complete epithelin cDNAs were obtained from rat, mouse, and humansources by using a PCR protocol to isolate the 5' and 3' ends ofmessages that have a known central sequence, and by screening λgt10libraries. In particular, a PCR strategy with exact epithelin primersoriented in the 3' and 5' directions in combination with primers thatanneal to the natural poly(A) tail, or a synthetic poly(A) track addedonto the 5' extended cDNA was employed (Plowman et al., 1990, Proc.Natl. Acad. Sci. U.S.A. 87:4905-08). A rat kidney cDNA library wasconstructed in λgt10 (Plowman et al., 1990, Mol. Cell Biol. 10:1969-81),and a full length rat epithelin cDNA was isolated by screening 2.0×10⁵recombinants with PCR generated epithelin probes. These probes were alsoused to obtain the mouse epithelin gene from a mouse T--cell genomiclibrary (Stratagene, La Jolla, Calif.). Several PCR-generated clones,rat cDNA clones, and the mouse genomic clones were sequenced on bothstrands by using T7 polymerase with oligonucleotide primers (Tabor andRichardson 1987, Proc. Natl. Acad. Sci. U.S.A. 84:4767-71.

The composite sequence of the rat epithelin cDNA (SEQ ID NO: 1) is 2150bp long, which closely approximates the 2.3 kilobase transcript seen bynorthern analysis with rat kidney mRNA. The sequence predicts a589-residue preprotein with a 30 bp 5'-untranslated region and a 343 bp3'-untranslated region. The first AUG is followed by an N-terminalhydrophobic signal peptide of 16 amino acids and the 573 amino acidmature epithelin precursor with a predicted M_(r) of 61,597. Theprecursor has no transmembrane domain, 3 potential N-linkedglycosylation sites, and 88 cysteine residues. The epithelin precursorhas a highly repetitive organization (FIG. 18) containing 7 tandemcopies of a 55-57 amino acid consensus motif: VXCX₅₋₆ CX₅ CCXx₈ CCX₆CCXDX₂ HCCPX₄ CX₅₋₆ CX₂. Two of the repeats represent the known activemolecules, epithelin 1 and 2. The predicted amino acid sequences of themouse and human epithelin precursors contain 589 and 593 residues,respectively, and exhibit 86% (mouse) and 75% (human) sequence identitywith the rat protein (FIG. 20A). Additional isoforms of epithelin mayalso exist, since one rat cDNA clone has a deletion of 234 bp (FIG. 20A,amino acids 398-475), maintained the reading frame, and generated achimeric motif. This deletion is likely the result of alternativesplicing since its boundaries map precisely to the location ofexon-intron junctions in the mouse epithelin gene.

Comparison of the epithelin sequence with available protein and DNAsequence databases reveals three regions with homology to knownproteins. The first 41 amino acids following the signal sequence of theepithelin precursor contains 6 cysteine residues arranged in a patternsimilar to the N-terminal half of the other seven cystein-rich motifs.The sequence CPDGQFCPVACC is completely conserved between human, rat andmouse epithelins, and conforms to a consensus pattern present in afamily of snake toxins (Dufton, 1984, J. Mol. Evol. 20:128-34). A secondregion of homology exists within three of the cystein-rich motifs of ratepithelin (CCX₂ HX₂ C), and conforms to a consensus surrounding anactive site of phospholipase A2 (Gomez et al., 1989, J. Eur. J. Biochem.186:23-33). Finally, an extended homology exists between the 12 cysteinemotif of epithelin and the C-terminal regulatory domain of a tomatothiol protease (FIG. 20D). This noncatalytic domain has beenhypothesized to regulate the protease activity by binding to heavymetals. The alternating cysteine and histidine residues in the epithelinprecursor is reminiscent of metal-binding domains of a variety ofproteins, although the epithelin motif does not conform to that of anyknown metal-binding consensus. Northern analysis demonstrates that the2.3 kilobase epithelin transcript is ubiquitously expressed, and ispredominant in the adult kidney, placenta, heart, duodenum, colon, andcerebral cortex. In addition, it is present in a wide variety ofepithelial tumor cell lines. Southern analysis indicates that theepithelin precursor is encoded by a single copy gene. These resultssuggest a post-transcriptional mechanism for the generation of activemolecules from the epithelin precursor.

7.2. EXPRESSION IN COS CELLS

The biochemical properties of rat epithelin was determined by insertingits complete coding sequence into an expression vector under the controlof the cytomegalovirus immediate-early promoter (Seed and Aruffo, 1987,Proc. Natl. Acad. Sci. U.S.A. 84:3365-69). Specifically, a 1.6 kbfragment containing the complete rat epithelin coding sequence wasinserted into a cDM8 based expression vector, generating the expressionvector crEPN1.6. A similar construct, crEPN1.4, was prepared byinsertion of a 1.35 kb fragment from an epithelin cDNA that has a singleexon deletion, thereby eliminating amino acids 398-475. The secretionplasmids, cβrEPN1 and cβrEPN2, were constructed by ligating a syntheticsimian TGF-β1 signal sequence (SEQ ID NO: 11) contained on theoligonucleotides

    __________________________________________________________________________                                   M  P  P  S  G  L  R  L  L  P  L  L              5'40  AGCTTCTGCAGGGGCGGGGCCTCCCCC ATGCCGCCCTCCGGGCTGCGGCTGCTGCCGCTGCTG        3'40      AGACGTCCCCGCCCCGGAGGGGG TACGGCGGGAGGCCCGACGCCGACGACGGCGACGAC         -    L  P  L  L  W  L  L  V  L  T  P  S  R  P  A  A                             CTACCGCTGCTGTGGCTACTGGTGCTGACGCCTAGCCGGCCGGCCGC 3'                            GATGGCGACGACACCGATGACCACGACTGCGGATCGGCCGGCCGG   5'                        __________________________________________________________________________

and PCR generated SacII-XbaI fragments containing the coding sequence ofepithelin 1 and 2 into the cDM8SII plasmid digested with HindIII andXbaI. cDM8SII is a modified cDM8 vector, where the unique SacII site hasbeen eliminated with Klenow. The new SacII site places a final glycineof the signal peptide in frame with the epithelin coding sequence. Theexpression plasmids were grown in competent MC1061/P3 bacteria, andintroduced into COS-1 cells using the DEAE-dextran method (Seed andAruffo, 1987, Proc. Natl. Acad. Sci. U.S.A. 84:3365-69). Forty-eighthours after transfection, the cells were washed with phosphate bufferedsaline, and labeled for 16 hours in serum free MEM (4 ml per 100 mmplate) supplemented with 25 μCi/ml ³⁵ S-cysteine (1100 Ci/mmole, NewEngland Nuclear). Labeled supernatants were dialyzed against 0.1 Nacetic acid, dried, and 1 ml equivalents run on 10% or 15%SDS-polyacrylamide gels.

The transiently expressed protein was readily detected by SDS-PAGEanalysis of ³⁵ S-cysteine labeled supernatants. The recombinant proteinhad a molecular mass of about 75K, slightly more than the predicted 62K,possibly due to glycosylation (FIG. 21A, lane 1). A similar constructencoding an epithelin isoform, but lacking an exon in the C-terminalregion, migrated with average M_(r) of 68K, commensurate with thedeletion of 78 amino acids. There was no evidence of the precursor beingprocessed into smaller forms. To express the mature recombinantepithelin 1 and 2 proteins, their coding regions were placed into anexpression vector behind a synthetic signal peptide sequence (cβrEPN1and cβrEPN2, respectively), which resulted in secretion of the 6Kproteins from COS cells (FIG. 21B). The cells transfected with theseconstructs were growth inhibited and exhibited an altered morphologywith many cells showing a signet ring appearance, compared with theintact monolayer seen on a mock transfection. Supernatant from thecβrEPN1 transfection was partially purified on a Bio-Sil TSK-250 column(Shoyab et al., 1990, 87:4905-09) and fractions were assayed for (growthinhibitory activity (GIA) on A431 cells. These cells secreted activeepithelin 1 (approximately 25 GIA units/100 mm plate) whereas thesupernatant from mock transfected cells had no detectable activity (oneGIA unit corresponds to the amount of material required for 50%inhibition of cell growth).

Epithelin 1 has an inhibitory effect on A431 cells and it acts asmitogen on several normal epithelial cell lines (See Section 6., supra).In addition, epithelin 1 can induce anchorage-independent growth of ratkidney fibroblasts in the presence of transforming growth factor β (FIG.17). In contrast, epithelin 2 has no effect on the growth of rodentkeratinocytes on fibroblasts and it opposed the mitogenic effects ofepithelin 1 (in a dose dependent manner) in both of these systems (FIG.17). The unprocessed epithelin precursor had no activity in any of theseassays. Perhaps the intact precursor serves an entirely different rolethan the processed forms, such as chelating metal ions, or regulation ofproteases and phospholipases.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 12                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1767 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1767                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG TGG ATC CTG GTG AGC TGG CTG GCC TTA GT - #G GCA AGG CTG GTG GCT           48                                                                       Met Trp Ile Leu Val Ser Trp Leu Ala Leu Va - #l Ala Arg Leu Val Ala             1               5 - #                 10 - #                 15              - - GGA ACA CAG TGC CCA GAT GGT CAA TTC TGC CC - #T GTT GCC TGC TGC CTT           96                                                                       Gly Thr Gln Cys Pro Asp Gly Gln Phe Cys Pr - #o Val Ala Cys Cys Leu                        20     - #             25     - #             30                  - - GAC CAG GGA GGA GCC AAC TAC AGC TGC TGT AA - #C CCT CTT CTG GAC ACA          144                                                                       Asp Gln Gly Gly Ala Asn Tyr Ser Cys Cys As - #n Pro Leu Leu Asp Thr                    35         - #         40         - #         45                      - - TGG CCT ATA ATA ACG AGC CGT CGT CTA GAT GG - #C TCC TGC CAG ATC CGT          192                                                                       Trp Pro Ile Ile Thr Ser Arg Arg Leu Asp Gl - #y Ser Cys Gln Ile Arg                50             - #     55             - #     60                          - - GAC CAC TGT CCT GAT GGC TAC TCT TGT CTT CT - #C ACT GTG TCT GGG ACT          240                                                                       Asp His Cys Pro Asp Gly Tyr Ser Cys Leu Le - #u Thr Val Ser Gly Thr            65                 - # 70                 - # 75                 - # 80       - - TCC AGC TGC TGC CCG TTC TCT GAG GGT GTA TC - #T TGT GAT GAT GGC CAG          288                                                                       Ser Ser Cys Cys Pro Phe Ser Glu Gly Val Se - #r Cys Asp Asp Gly Gln                            85 - #                 90 - #                 95              - - CAC TGC TGC CCC CGG GGC TTC CAC TGT AGT GC - #G GAT GGG AAA TCC TGC          336                                                                       His Cys Cys Pro Arg Gly Phe His Cys Ser Al - #a Asp Gly Lys Ser Cys                       100      - #           105      - #           110                  - - TCT CAG ATA TCA GAT AGC CTC TTG GGT GCT GT - #C CAG TGT CCT GGT AGC          384                                                                       Ser Gln Ile Ser Asp Ser Leu Leu Gly Ala Va - #l Gln Cys Pro Gly Ser                   115          - #       120          - #       125                      - - CAG TTC GAA TGT CCT GAC TCC GCC ACC TGC TG - #T ATT ATG ATT GAT GGT          432                                                                       Gln Phe Glu Cys Pro Asp Ser Ala Thr Cys Cy - #s Ile Met Ile Asp Gly               130              - #   135              - #   140                          - - TCC TGG GGG TGC TGC CCC ATG CCC CAG GCC TC - #T TGC TGT GAA GAC AGA          480                                                                       Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Se - #r Cys Cys Glu Asp Arg           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GTG CAT TGC TGT CCC CAC GGG GCC TCC TGT GA - #C CTG GTT CAC ACG        CGA      528                                                                    Val His Cys Cys Pro His Gly Ala Ser Cys As - #p Leu Val His Thr Arg                          165  - #               170  - #               175              - - TGC ATT TCA CCC ACG GGC ACC CAC CCC TTA CT - #A AAG AAA TTC CCC GCA          576                                                                       Cys Ile Ser Pro Thr Gly Thr His Pro Leu Le - #u Lys Lys Phe Pro Ala                       180      - #           185      - #           190                  - - CAA AGG ACC AAC AGG GCA GTG GCT TTC CCT TT - #T TCC GTG GTG TGC CCT          624                                                                       Gln Arg Thr Asn Arg Ala Val Ala Phe Pro Ph - #e Ser Val Val Cys Pro                   195          - #       200          - #       205                      - - GAT GCT AAG ACC CAG TGC CCT GAT GAC TCT AC - #C TGC TGT GAG CTA CCC          672                                                                       Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Th - #r Cys Cys Glu Leu Pro               210              - #   215              - #   220                          - - ACT GGG AAG TAT GGC TGT TGT CCA ATG CCC AA - #C GCC ATC TGC TGT TCC          720                                                                       Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro As - #n Ala Ile Cys Cys Ser           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GAC CAC CTG CAC TGC TGC CCC CAG GAC ACT GT - #A TGT GAC CTG ATC        CAG      768                                                                    Asp His Leu His Cys Cys Pro Gln Asp Thr Va - #l Cys Asp Leu Ile Gln                          245  - #               250  - #               255              - - AGC AAG TGC ATA TCC AAG GAC TAC ACC ACA GA - #T CTC ATG ACC AAG CTG          816                                                                       Ser Lys Cys Ile Ser Lys Asp Tyr Thr Thr As - #p Leu Met Thr Lys Leu                       260      - #           265      - #           270                  - - CCT GGA TAC CCA GTG AAT GAG GTG AAG TGC GA - #C TTG GAG GTG AGC TGT          864                                                                       Pro Gly Tyr Pro Val Asn Glu Val Lys Cys As - #p Leu Glu Val Ser Cys                   275          - #       280          - #       285                      - - CCT GAT GGC TAC ACC TGC TGC CGC CTC AAC AC - #T GGG GCC TGG GGC TGC          912                                                                       Pro Asp Gly Tyr Thr Cys Cys Arg Leu Asn Th - #r Gly Ala Trp Gly Cys               290              - #   295              - #   300                          - - TGT CCA TTC ACC AAG GCT GTG TGT TGT GAA GA - #C CAC ATT CAC TGC TGC          960                                                                       Cys Pro Phe Thr Lys Ala Val Cys Cys Glu As - #p His Ile His Cys Cys           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - CCA GCC GGG TTT CAG TGT CAC ACA GAG ACA GG - #A ACC TGT GAA CTG        GGA     1008                                                                    Pro Ala Gly Phe Gln Cys His Thr Glu Thr Gl - #y Thr Cys Glu Leu Gly                          325  - #               330  - #               335              - - GTC CTT CAG GTA CCC TGG ATG AAA AAG GTC AC - #G GCC TCC CTC AGC CTG         1056                                                                       Val Leu Gln Val Pro Trp Met Lys Lys Val Th - #r Ala Ser Leu Ser Leu                       340      - #           345      - #           350                  - - CCA GAC CCA CAG ATC TTG AAG AAT GAT GTC CC - #C TGT GAT GAC TTC AGT         1104                                                                       Pro Asp Pro Gln Ile Leu Lys Asn Asp Val Pr - #o Cys Asp Asp Phe Ser                   355          - #       360          - #       365                      - - AGC TGT CCT TCT AAC AAT ACC TGC TGC AGA CT - #C AGT TCT GGG GAC TGG         1152                                                                       Ser Cys Pro Ser Asn Asn Thr Cys Cys Arg Le - #u Ser Ser Gly Asp Trp               370              - #   375              - #   380                          - - GGC TGC TGT CCC ATC CCA GAG GCT GTC TGC TG - #C TTA GAC CAC CAG CAT         1200                                                                       Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cy - #s Leu Asp His Gln His           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - TGC TGC CCT CAG GGT TTC AAA TGT ATG GAT GA - #G GGG TAC TGT CAG        AAG     1248                                                                    Cys Cys Pro Gln Gly Phe Lys Cys Met Asp Gl - #u Gly Tyr Cys Gln Lys                          405  - #               410  - #               415              - - GGA GAC AGA ATG GTG GCT GGC CTG GAG AAG AT - #G CCT GTC CGC CAG ACA         1296                                                                       Gly Asp Arg Met Val Ala Gly Leu Glu Lys Me - #t Pro Val Arg Gln Thr                       420      - #           425      - #           430                  - - ACT CTG CTC CAA CAT GGA GAT ATT GGT TGT GA - #C CAG CAT ACC AGC TGC         1344                                                                       Thr Leu Leu Gln His Gly Asp Ile Gly Cys As - #p Gln His Thr Ser Cys                   435          - #       440          - #       445                      - - CCA GTA GGG CAA ACA TGC TGC CCA AGC CTG AA - #G GGA AGT TGG GCC TGC         1392                                                                       Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Ly - #s Gly Ser Trp Ala Cys               450              - #   455              - #   460                          - - TGC CAG TTG CCC CAT GCT GTG TGC TGT GAG GA - #C CGG CAG CAC TGT TGC         1440                                                                       Cys Gln Leu Pro His Ala Val Cys Cys Glu As - #p Arg Gln His Cys Cys           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - CCG GCT GGG TAC ACC TGC AAC GTG AAG GCG AG - #A ACC TGT GAG AAG        GAT     1488                                                                    Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Ar - #g Thr Cys Glu Lys Asp                          485  - #               490  - #               495              - - GCA GGC TCT GTC CAG CCT TCC ATG GAC CTG AC - #C TTT GGC TCT AAG GTT         1536                                                                       Ala Gly Ser Val Gln Pro Ser Met Asp Leu Th - #r Phe Gly Ser Lys Val                       500      - #           505      - #           510                  - - GGG AAT GTG GAA TGT GGT GCC GGA CAT TTC TG - #C CAT GAT AAC CAG TCC         1584                                                                       Gly Asn Val Glu Cys Gly Ala Gly His Phe Cy - #s His Asp Asn Gln Ser                   515          - #       520          - #       525                      - - TGT TGT AAA GAC AGC CAA GGA GGC TGG GCC TG - #C TGT CCC TAT GTA AAG         1632                                                                       Cys Cys Lys Asp Ser Gln Gly Gly Trp Ala Cy - #s Cys Pro Tyr Val Lys               530              - #   535              - #   540                          - - GGT GTC TGC TGT AGA GAT GGA CGT CAC TGT TG - #T CCC ATT GGC TTC CAC         1680                                                                       Gly Val Cys Cys Arg Asp Gly Arg His Cys Cy - #s Pro Ile Gly Phe His           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - TGT TCA GCC AAG GGA ACC AAG TGT TTG CGG AA - #G AAG ACC CCT CGC        TGG     1728                                                                    Cys Ser Ala Lys Gly Thr Lys Cys Leu Arg Ly - #s Lys Thr Pro Arg Trp                          565  - #               570  - #               575              - - GAC ATA CTT TTG AGG GAT CCA GCC CCA AGA CC - #G CTA CTG                  - #   1767                                                                    Asp Ile Leu Leu Arg Asp Pro Ala Pro Arg Pr - #o Leu Leu                                   580      - #           585                                         - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 589 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Trp Ile Leu Val Ser Trp Leu Ala Leu Va - #l Ala Arg Leu Val Ala        1               5 - #                 10 - #                 15              - - Gly Thr Gln Cys Pro Asp Gly Gln Phe Cys Pr - #o Val Ala Cys Cys Leu                   20     - #             25     - #             30                  - - Asp Gln Gly Gly Ala Asn Tyr Ser Cys Cys As - #n Pro Leu Leu Asp Thr               35         - #         40         - #         45                      - - Trp Pro Ile Ile Thr Ser Arg Arg Leu Asp Gl - #y Ser Cys Gln Ile Arg           50             - #     55             - #     60                          - - Asp His Cys Pro Asp Gly Tyr Ser Cys Leu Le - #u Thr Val Ser Gly Thr       65                 - # 70                 - # 75                 - # 80       - - Ser Ser Cys Cys Pro Phe Ser Glu Gly Val Se - #r Cys Asp Asp Gly Gln                       85 - #                 90 - #                 95              - - His Cys Cys Pro Arg Gly Phe His Cys Ser Al - #a Asp Gly Lys Ser Cys                  100      - #           105      - #           110                  - - Ser Gln Ile Ser Asp Ser Leu Leu Gly Ala Va - #l Gln Cys Pro Gly Ser              115          - #       120          - #       125                      - - Gln Phe Glu Cys Pro Asp Ser Ala Thr Cys Cy - #s Ile Met Ile Asp Gly          130              - #   135              - #   140                          - - Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Se - #r Cys Cys Glu Asp Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val His Cys Cys Pro His Gly Ala Ser Cys As - #p Leu Val His Thr        Arg                                                                                             165  - #               170  - #               175             - - Cys Ile Ser Pro Thr Gly Thr His Pro Leu Le - #u Lys Lys Phe Pro Ala                  180      - #           185      - #           190                  - - Gln Arg Thr Asn Arg Ala Val Ala Phe Pro Ph - #e Ser Val Val Cys Pro              195          - #       200          - #       205                      - - Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Th - #r Cys Cys Glu Leu Pro          210              - #   215              - #   220                          - - Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro As - #n Ala Ile Cys Cys Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp His Leu His Cys Cys Pro Gln Asp Thr Va - #l Cys Asp Leu Ile        Gln                                                                                             245  - #               250  - #               255             - - Ser Lys Cys Ile Ser Lys Asp Tyr Thr Thr As - #p Leu Met Thr Lys Leu                  260      - #           265      - #           270                  - - Pro Gly Tyr Pro Val Asn Glu Val Lys Cys As - #p Leu Glu Val Ser Cys              275          - #       280          - #       285                      - - Pro Asp Gly Tyr Thr Cys Cys Arg Leu Asn Th - #r Gly Ala Trp Gly Cys          290              - #   295              - #   300                          - - Cys Pro Phe Thr Lys Ala Val Cys Cys Glu As - #p His Ile His Cys Cys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Ala Gly Phe Gln Cys His Thr Glu Thr Gl - #y Thr Cys Glu Leu        Gly                                                                                             325  - #               330  - #               335             - - Val Leu Gln Val Pro Trp Met Lys Lys Val Th - #r Ala Ser Leu Ser Leu                  340      - #           345      - #           350                  - - Pro Asp Pro Gln Ile Leu Lys Asn Asp Val Pr - #o Cys Asp Asp Phe Ser              355          - #       360          - #       365                      - - Ser Cys Pro Ser Asn Asn Thr Cys Cys Arg Le - #u Ser Ser Gly Asp Trp          370              - #   375              - #   380                          - - Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cy - #s Leu Asp His Gln His      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Cys Cys Pro Gln Gly Phe Lys Cys Met Asp Gl - #u Gly Tyr Cys Gln        Lys                                                                                             405  - #               410  - #               415             - - Gly Asp Arg Met Val Ala Gly Leu Glu Lys Me - #t Pro Val Arg Gln Thr                  420      - #           425      - #           430                  - - Thr Leu Leu Gln His Gly Asp Ile Gly Cys As - #p Gln His Thr Ser Cys              435          - #       440          - #       445                      - - Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Ly - #s Gly Ser Trp Ala Cys          450              - #   455              - #   460                          - - Cys Gln Leu Pro His Ala Val Cys Cys Glu As - #p Arg Gln His Cys Cys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Ar - #g Thr Cys Glu Lys        Asp                                                                                             485  - #               490  - #               495             - - Ala Gly Ser Val Gln Pro Ser Met Asp Leu Th - #r Phe Gly Ser Lys Val                  500      - #           505      - #           510                  - - Gly Asn Val Glu Cys Gly Ala Gly His Phe Cy - #s His Asp Asn Gln Ser              515          - #       520          - #       525                      - - Cys Cys Lys Asp Ser Gln Gly Gly Trp Ala Cy - #s Cys Pro Tyr Val Lys          530              - #   535              - #   540                          - - Gly Val Cys Cys Arg Asp Gly Arg His Cys Cy - #s Pro Ile Gly Phe His      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Cys Ser Ala Lys Gly Thr Lys Cys Leu Arg Ly - #s Lys Thr Pro Arg        Trp                                                                                             565  - #               570  - #               575             - - Asp Ile Leu Leu Arg Asp Pro Ala Pro Arg Pr - #o Leu Leu                              580      - #           585                                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1779 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                                (F) TISSUE TYPE: Kidney                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1779                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - ATG TGG ACC CTG GTG AGC TGG GTG GCC TTA AC - #A GCA GGG CTG GTG GCT           48                                                                       Met Trp Thr Leu Val Ser Trp Val Ala Leu Th - #r Ala Gly Leu Val Ala             1               5 - #                 10 - #                 15              - - GGA ACG CGG TGC CCA GAT GGT CAG TTC TGC CC - #T GTG GCC TGC TGC CTG           96                                                                       Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pr - #o Val Ala Cys Cys Leu                        20     - #             25     - #             30                  - - GAC CCC GGA GGA GCC AGC TAC AGC TGC TGC CG - #T CCC CTT CTG GAC AAA          144                                                                       Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Ar - #g Pro Leu Leu Asp Lys                    35         - #         40         - #         45                      - - TGG CCC ACA ACA CTG AGC AGG CAT CTG GGT GG - #C CCC TGC CAG GTT GAT          192                                                                       Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gl - #y Pro Cys Gln Val Asp                50             - #     55             - #     60                          - - GCC CAC TGC TCT GCC GGC CAC TCC TGC ATC TT - #T ACC GTC TCA GGG ACT          240                                                                       Ala His Cys Ser Ala Gly His Ser Cys Ile Ph - #e Thr Val Ser Gly Thr            65                 - # 70                 - # 75                 - # 80       - - TCC AGT TGC TGC CCC TTC CCA GAG GCC GTG GC - #A TGC GGG GAT GGC CAT          288                                                                       Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Al - #a Cys Gly Asp Gly His                            85 - #                 90 - #                 95              - - CAC TGC TGC CCA CGG GGC TTC CAC TGC AGT GC - #A GAC GGG CGA TCC TGC          336                                                                       His Cys Cys Pro Arg Gly Phe His Cys Ser Al - #a Asp Gly Arg Ser Cys                       100      - #           105      - #           110                  - - TTC CAA AGA TCA GGT AAC AAC TCC GTG GGT GC - #C ATC CAG TGC CCT GAT          384                                                                       Phe Gln Arg Ser Gly Asn Asn Ser Val Gly Al - #a Ile Gln Cys Pro Asp                   115          - #       120          - #       125                      - - AGT CAG TTC GAA TGC CCG GAC TTC TCC ACG TG - #C TGT GTT ATG GTC GAT          432                                                                       Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cy - #s Cys Val Met Val Asp               130              - #   135              - #   140                          - - GGC TCC TGG GGG TGC TGC CCC ATG CCC CAG GC - #T TCC TGC TGT GAA GAC          480                                                                       Gly Ser Trp Gly Cys Cys Pro Met Pro Gln Al - #a Ser Cys Cys Glu Asp           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - AGG GTG CAC TGC TGT CCG CAC GGT GCC TTC TG - #C GAC CTG GTT CAC        ACC      528                                                                    Arg Val His Cys Cys Pro His Gly Ala Phe Cy - #s Asp Leu Val His Thr                          165  - #               170  - #               175              - - CGC TGC ATC ACA CCC ACG GGC ACC CAC CCC CT - #G GCA AAG AAG CTC CCT          576                                                                       Arg Cys Ile Thr Pro Thr Gly Thr His Pro Le - #u Ala Lys Lys Leu Pro                       180      - #           185      - #           190                  - - GCC CAG AGG ACT AAC AGG GCA GTG GCC TTG TC - #C AGC TCG GTC ATG TGT          624                                                                       Ala Gln Arg Thr Asn Arg Ala Val Ala Leu Se - #r Ser Ser Val Met Cys                   195          - #       200          - #       205                      - - CCG GAC GCA CGG TCC CGG TGC CCT GAT GGT TC - #T ACC TGC TGT GAG CTG          672                                                                       Pro Asp Ala Arg Ser Arg Cys Pro Asp Gly Se - #r Thr Cys Cys Glu Leu               210              - #   215              - #   220                          - - CCC AGT GGG AAG TAT GGC TGC TGC CCA ATG CC - #C AAC GCC ACC TGC TGC          720                                                                       Pro Ser Gly Lys Tyr Gly Cys Cys Pro Met Pr - #o Asn Ala Thr Cys Cys           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - TCC GAT CAC CTG CAC TGC TGC CCC CAA GAC AC - #T GTG TGT GAC CTG        ATC      768                                                                    Ser Asp His Leu His Cys Cys Pro Gln Asp Th - #r Val Cys Asp Leu Ile                          245  - #               250  - #               255              - - CAG AGT AAG TGC CTC TCC AAG GAG AAC GCT AC - #C ACG GAC CTC CTC ACT          816                                                                       Gln Ser Lys Cys Leu Ser Lys Glu Asn Ala Th - #r Thr Asp Leu Leu Thr                       260      - #           265      - #           270                  - - AAG CTG CCT GCG CAC ACA GTG GGG GAT GTG AA - #A TGT GAC ATG GAG GTG          864                                                                       Lys Leu Pro Ala His Thr Val Gly Asp Val Ly - #s Cys Asp Met Glu Val                   275          - #       280          - #       285                      - - AGC TGC CCA GAT GGC TAT ACC TGC TGC CGT CT - #A CAG TCG GGG GCC TGG          912                                                                       Ser Cys Pro Asp Gly Tyr Thr Cys Cys Arg Le - #u Gln Ser Gly Ala Trp               290              - #   295              - #   300                          - - GGC TGC TGC CCT TTT ACC CAG GCT GTG TGC TG - #T GAG GAC CAC ATA CAC          960                                                                       Gly Cys Cys Pro Phe Thr Gln Ala Val Cys Cy - #s Glu Asp His Ile His           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - TGC TGT CCC GCG GGG TTT ACG TGT GAC ACG CA - #G AAG GGT ACC TGT        GAA     1008                                                                    Cys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gl - #n Lys Gly Thr Cys Glu                          325  - #               330  - #               335              - - CAG GGG CCC CAC CAG GTG CCC TGG ATG GAG AA - #G GCC CCA GCT CAC CTC         1056                                                                       Gln Gly Pro His Gln Val Pro Trp Met Glu Ly - #s Ala Pro Ala His Leu                       340      - #           345      - #           350                  - - AGC CTG CCA GAC CCA CAA GCC TTG AAG AGA GA - #T GTC CCC TGT GAT AAT         1104                                                                       Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg As - #p Val Pro Cys Asp Asn                   355          - #       360          - #       365                      - - GTC AGC AGC TGT CCC TCC TCC GAT ACC TGC TG - #C CAA CTC ACG TCT GGG         1152                                                                       Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cy - #s Gln Leu Thr Ser Gly               370              - #   375              - #   380                          - - GAG TGG GGC TGC TGT CCA ATC CCA GAG GCT GT - #C TGC TGC TCG GAC CAC         1200                                                                       Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Va - #l Cys Cys Ser Asp His           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - CAG CAC TGC TGC CCC CAG GGC TAC ACG TGT GT - #A GCT GAG GGG CAG        TGT     1248                                                                    Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Va - #l Ala Glu Gly Gln Cys                          405  - #               410  - #               415              - - CAG CGA GGA AGC GAG ATC GTG GCT GGA CTG GA - #G AAG ATG CCT GCC CGC         1296                                                                       Gln Arg Gly Ser Glu Ile Val Ala Gly Leu Gl - #u Lys Met Pro Ala Arg                       420      - #           425      - #           430                  - - CGG GCT TCC TTA TCC CAC CCC AGA GAC ATC GG - #C TGT GAC CAG CAC ACC         1344                                                                       Arg Ala Ser Leu Ser His Pro Arg Asp Ile Gl - #y Cys Asp Gln His Thr                   435          - #       440          - #       445                      - - AGC TGC CCG GTG GGG CAG ACC TGC TGC CCG AG - #C CTG GGT GGG AGC TGG         1392                                                                       Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Se - #r Leu Gly Gly Ser Trp               450              - #   455              - #   460                          - - GCC TGC TGC CAG TTG CCC CAT GCT GTG TGC TG - #C GAG GAT CGC CAG CAC         1440                                                                       Ala Cys Cys Gln Leu Pro His Ala Val Cys Cy - #s Glu Asp Arg Gln His           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - TGC TGC CCG GCT GGC TAC ACC TGC AAC GTG AA - #G GCT CGA TCC TGC        GAG     1488                                                                    Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Ly - #s Ala Arg Ser Cys Glu                          485  - #               490  - #               495              - - AAG GAA GTG GTC TCT GCC CAG CCT GCC ACC TT - #C CTG GCC CGT AGC CCT         1536                                                                       Lys Glu Val Val Ser Ala Gln Pro Ala Thr Ph - #e Leu Ala Arg Ser Pro                       500      - #           505      - #           510                  - - CAC GTG GGT GTG AAG GAC GTG GAG TGT GGG GA - #A GGA CAC TTC TGC CAT         1584                                                                       His Val Gly Val Lys Asp Val Glu Cys Gly Gl - #u Gly His Phe Cys His                   515          - #       520          - #       525                      - - GAT AAC CAG ACC TGC TGC CGA GAC AAC CGA CA - #G GGC TGG GCC TGC TGT         1632                                                                       Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gl - #n Gly Trp Ala Cys Cys               530              - #   535              - #   540                          - - CCC TAC CGC CAG GGC GTC TGT TGT GCT GAT CG - #G CGC CAC TGC TGT CCT         1680                                                                       Pro Tyr Arg Gln Gly Val Cys Cys Ala Asp Ar - #g Arg His Cys Cys Pro           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - GCT GGC TTC CGC TGC GCA GCC AGG GGT ACC AA - #G TGT TTG CGC AGG        GAG     1728                                                                    Ala Gly Phe Arg Cys Ala Ala Arg Gly Thr Ly - #s Cys Leu Arg Arg Glu                          565  - #               570  - #               575              - - GCC CCG CGC TGG GAC GCC CCT TTG AGG GAC CC - #A GCC TTG AGA CAG CTG         1776                                                                       Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pr - #o Ala Leu Arg Gln Leu                       580      - #           585      - #           590                  - - CTG                  - #                  - #                  - #               1779                                                                  Leu                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 593 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Trp Thr Leu Val Ser Trp Val Ala Leu Th - #r Ala Gly Leu Val Ala        1               5 - #                 10 - #                 15              - - Gly Thr Arg Cys Pro Asp Gly Gln Phe Cys Pr - #o Val Ala Cys Cys Leu                   20     - #             25     - #             30                  - - Asp Pro Gly Gly Ala Ser Tyr Ser Cys Cys Ar - #g Pro Leu Leu Asp Lys               35         - #         40         - #         45                      - - Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gl - #y Pro Cys Gln Val Asp           50             - #     55             - #     60                          - - Ala His Cys Ser Ala Gly His Ser Cys Ile Ph - #e Thr Val Ser Gly Thr       65                 - # 70                 - # 75                 - # 80       - - Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Al - #a Cys Gly Asp Gly His                       85 - #                 90 - #                 95              - - His Cys Cys Pro Arg Gly Phe His Cys Ser Al - #a Asp Gly Arg Ser Cys                  100      - #           105      - #           110                  - - Phe Gln Arg Ser Gly Asn Asn Ser Val Gly Al - #a Ile Gln Cys Pro Asp              115          - #       120          - #       125                      - - Ser Gln Phe Glu Cys Pro Asp Phe Ser Thr Cy - #s Cys Val Met Val Asp          130              - #   135              - #   140                          - - Gly Ser Trp Gly Cys Cys Pro Met Pro Gln Al - #a Ser Cys Cys Glu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Val His Cys Cys Pro His Gly Ala Phe Cy - #s Asp Leu Val His        Thr                                                                                             165  - #               170  - #               175             - - Arg Cys Ile Thr Pro Thr Gly Thr His Pro Le - #u Ala Lys Lys Leu Pro                  180      - #           185      - #           190                  - - Ala Gln Arg Thr Asn Arg Ala Val Ala Leu Se - #r Ser Ser Val Met Cys              195          - #       200          - #       205                      - - Pro Asp Ala Arg Ser Arg Cys Pro Asp Gly Se - #r Thr Cys Cys Glu Leu          210              - #   215              - #   220                          - - Pro Ser Gly Lys Tyr Gly Cys Cys Pro Met Pr - #o Asn Ala Thr Cys Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Asp His Leu His Cys Cys Pro Gln Asp Th - #r Val Cys Asp Leu        Ile                                                                                             245  - #               250  - #               255             - - Gln Ser Lys Cys Leu Ser Lys Glu Asn Ala Th - #r Thr Asp Leu Leu Thr                  260      - #           265      - #           270                  - - Lys Leu Pro Ala His Thr Val Gly Asp Val Ly - #s Cys Asp Met Glu Val              275          - #       280          - #       285                      - - Ser Cys Pro Asp Gly Tyr Thr Cys Cys Arg Le - #u Gln Ser Gly Ala Trp          290              - #   295              - #   300                          - - Gly Cys Cys Pro Phe Thr Gln Ala Val Cys Cy - #s Glu Asp His Ile His      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Cys Cys Pro Ala Gly Phe Thr Cys Asp Thr Gl - #n Lys Gly Thr Cys        Glu                                                                                             325  - #               330  - #               335             - - Gln Gly Pro His Gln Val Pro Trp Met Glu Ly - #s Ala Pro Ala His Leu                  340      - #           345      - #           350                  - - Ser Leu Pro Asp Pro Gln Ala Leu Lys Arg As - #p Val Pro Cys Asp Asn              355          - #       360          - #       365                      - - Val Ser Ser Cys Pro Ser Ser Asp Thr Cys Cy - #s Gln Leu Thr Ser Gly          370              - #   375              - #   380                          - - Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Va - #l Cys Cys Ser Asp His      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Va - #l Ala Glu Gly Gln        Cys                                                                                             405  - #               410  - #               415             - - Gln Arg Gly Ser Glu Ile Val Ala Gly Leu Gl - #u Lys Met Pro Ala Arg                  420      - #           425      - #           430                  - - Arg Ala Ser Leu Ser His Pro Arg Asp Ile Gl - #y Cys Asp Gln His Thr              435          - #       440          - #       445                      - - Ser Cys Pro Val Gly Gln Thr Cys Cys Pro Se - #r Leu Gly Gly Ser Trp          450              - #   455              - #   460                          - - Ala Cys Cys Gln Leu Pro His Ala Val Cys Cy - #s Glu Asp Arg Gln His      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Cys Cys Pro Ala Gly Tyr Thr Cys Asn Val Ly - #s Ala Arg Ser Cys        Glu                                                                                             485  - #               490  - #               495             - - Lys Glu Val Val Ser Ala Gln Pro Ala Thr Ph - #e Leu Ala Arg Ser Pro                  500      - #           505      - #           510                  - - His Val Gly Val Lys Asp Val Glu Cys Gly Gl - #u Gly His Phe Cys His              515          - #       520          - #       525                      - - Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gl - #n Gly Trp Ala Cys Cys          530              - #   535              - #   540                          - - Pro Tyr Arg Gln Gly Val Cys Cys Ala Asp Ar - #g Arg His Cys Cys Pro      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Ala Gly Phe Arg Cys Ala Ala Arg Gly Thr Ly - #s Cys Leu Arg Arg        Glu                                                                                             565  - #               570  - #               575             - - Ala Pro Arg Trp Asp Ala Pro Leu Arg Asp Pr - #o Ala Leu Arg Gln Leu                  580      - #           585      - #           590                  - - Leu                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1767 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mus muscu - #lus                                                (F) TISSUE TYPE: Kidney                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1767                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - ATG TGG GTC CTG ATG AGC TGG CTG GCC TTC GC - #G GCA GGG CTG GTA GCC           48                                                                       Met Trp Val Leu Met Ser Trp Leu Ala Phe Al - #a Ala Gly Leu Val Ala             1               5 - #                 10 - #                 15              - - GGA ACA CAG TGT CCA GAT GGG CAG TTC TGC CC - #T GTT GCC TGC TGC CTT           96                                                                       Gly Thr Gln Cys Pro Asp Gly Gln Phe Cys Pr - #o Val Ala Cys Cys Leu                        20     - #             25     - #             30                  - - GAC CAG GGA GGA GCC AAC TAC AGC TGC TGT AA - #C CCT CTT CTG GAC ACA          144                                                                       Asp Gln Gly Gly Ala Asn Tyr Ser Cys Cys As - #n Pro Leu Leu Asp Thr                    35         - #         40         - #         45                      - - TGG CCT AGA ATA ACG AGC CAT CAT CTA GAT GG - #C TCC TGC CAG ACC CAT          192                                                                       Trp Pro Arg Ile Thr Ser His His Leu Asp Gl - #y Ser Cys Gln Thr His                50             - #     55             - #     60                          - - GGC CAC TGT CCT GCT GGC TAT TCT TGT CTT CT - #C ACT GTG TCT GGG ACT          240                                                                       Gly His Cys Pro Ala Gly Tyr Ser Cys Leu Le - #u Thr Val Ser Gly Thr            65                 - # 70                 - # 75                 - # 80       - - TCC AGC TGC TGC CCG TTC TCT AAG GGT GTG TC - #T TGT GGT GAT GGC TAC          288                                                                       Ser Ser Cys Cys Pro Phe Ser Lys Gly Val Se - #r Cys Gly Asp Gly Tyr                            85 - #                 90 - #                 95              - - CAC TGC TGC CCC CAG GGC TTC CAC TGT AGT GC - #A GAT GGG AAA TCC TGC          336                                                                       His Cys Cys Pro Gln Gly Phe His Cys Ser Al - #a Asp Gly Lys Ser Cys                       100      - #           105      - #           110                  - - TTC CAG ATG TCA GAT AAC CCC TTG GGT GCT GT - #C CAG TGT CCT GGG AGC          384                                                                       Phe Gln Met Ser Asp Asn Pro Leu Gly Ala Va - #l Gln Cys Pro Gly Ser                   115          - #       120          - #       125                      - - CAG TTT GAA TGT CCT GAC TCT GCC ACC TGC TG - #C ATT ATG GTT GAT GGT          432                                                                       Gln Phe Glu Cys Pro Asp Ser Ala Thr Cys Cy - #s Ile Met Val Asp Gly               130              - #   135              - #   140                          - - TCG TGG GGA TGT TGT CCC ATG CCC CAG GCC TC - #T TGC TGT GAA GAC AGA          480                                                                       Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Se - #r Cys Cys Glu Asp Arg           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GTG CAT TGC TGT CCC CAT GGG GCC TCC TGT GA - #C CTG GTT CAC ACA        CGA      528                                                                    Val His Cys Cys Pro His Gly Ala Ser Cys As - #p Leu Val His Thr Arg                          165  - #               170  - #               175              - - TGC GTT TCA CCC ACG GGC ACC CAC ACC CTA CT - #A AAG AAG TTC CCT GCA          576                                                                       Cys Val Ser Pro Thr Gly Thr His Thr Leu Le - #u Lys Lys Phe Pro Ala                       180      - #           185      - #           190                  - - CAA AAG ACC AAC AGG GCA GTG TCT TTG CCT TT - #T TCT GTC GTG TGC CCT          624                                                                       Gln Lys Thr Asn Arg Ala Val Ser Leu Pro Ph - #e Ser Val Val Cys Pro                   195          - #       200          - #       205                      - - GAT GCT AAG ACC CAG TGT CCC GAT GAT TCT AC - #C TGC TGT GAG CTA CCC          672                                                                       Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Th - #r Cys Cys Glu Leu Pro               210              - #   215              - #   220                          - - ACT GGG AAG TAT GGC TGC TGT CCA ATG CCC AA - #T GCC ATC TGC TGT TCC          720                                                                       Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro As - #n Ala Ile Cys Cys Ser           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GAC CAC CTG CAC TGC TGC CCC CAG GAC ACT GT - #A TGT GAC CTG ATC        CAG      768                                                                    Asp His Leu His Cys Cys Pro Gln Asp Thr Va - #l Cys Asp Leu Ile Gln                          245  - #               250  - #               255              - - AGT AAG TGC CTA TCC AAG AAC TAC ACC ACG GA - #T CTC CTG ACC AAG CTG          816                                                                       Ser Lys Cys Leu Ser Lys Asn Tyr Thr Thr As - #p Leu Leu Thr Lys Leu                       260      - #           265      - #           270                  - - CCT GGA TAC CCA GTG AAG GAG GTG AAG TGC GA - #C ATG GAG GTG AGC TGC          864                                                                       Pro Gly Tyr Pro Val Lys Glu Val Lys Cys As - #p Met Glu Val Ser Cys                   275          - #       280          - #       285                      - - CCT GAA GGA TAT ACC TGC TGC CGC CTC AAC AC - #T GGG GCC TGG GGC TGC          912                                                                       Pro Glu Gly Tyr Thr Cys Cys Arg Leu Asn Th - #r Gly Ala Trp Gly Cys               290              - #   295              - #   300                          - - TGT CCA TTT GCC AAG GCC GTG TGT TGT GAG GA - #T CAC ATT CAT TGC TGC          960                                                                       Cys Pro Phe Ala Lys Ala Val Cys Cys Glu As - #p His Ile His Cys Cys           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - CCG GCA GGG TTT CAG TGT CAC ACA GAG AAA GG - #A ACC TGC GAA ATG        GGT     1008                                                                    Pro Ala Gly Phe Gln Cys His Thr Glu Lys Gl - #y Thr Cys Glu Met Gly                          325  - #               330  - #               335              - - ATC CTC CAA GTA CCC TGG ATG AAG AAG GTC AT - #A GCC CCC CGC CGC CTG         1056                                                                       Ile Leu Gln Val Pro Trp Met Lys Lys Val Il - #e Ala Pro Arg Arg Leu                       340      - #           345      - #           350                  - - CCA GAC CCA CAG ATC TTG AAG AGT GAT ACA CC - #T TGT GAT GAC TTC ACT         1104                                                                       Pro Asp Pro Gln Ile Leu Lys Ser Asp Thr Pr - #o Cys Asp Asp Phe Thr                   355          - #       360          - #       365                      - - AGG TGT CCT ACA AAC AAT ACC TGC TGC AAA CT - #C AAT TCT GGG GAC TGG         1152                                                                       Arg Cys Pro Thr Asn Asn Thr Cys Cys Lys Le - #u Asn Ser Gly Asp Trp               370              - #   375              - #   380                          - - GGC TGC TGT CCC ATC CCA GAG GCT GTC TGC TG - #C TCA GAC AAC CAG CAT         1200                                                                       Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cy - #s Ser Asp Asn Gln His           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - TGC TGC CCT CAG GGC TTC ACA TGT CTG GCT CA - #G GGG TAC TGT CAG        AAG     1248                                                                    Cys Cys Pro Gln Gly Phe Thr Cys Leu Ala Gl - #n Gly Tyr Cys Gln Lys                          405  - #               410  - #               415              - - GGA GAC ACA ATG GTG GCT GGC CTG GAG AAG AT - #A CCT GCC CGC CAG ACA         1296                                                                       Gly Asp Thr Met Val Ala Gly Leu Glu Lys Il - #e Pro Ala Arg Gln Thr                       420      - #           425      - #           430                  - - ACC CCG CTC CAA ATT GGA GAT ATC GGT TGT GA - #C CAG CAT ACC AGC TGC         1344                                                                       Thr Pro Leu Gln Ile Gly Asp Ile Gly Cys As - #p Gln His Thr Ser Cys                   435          - #       440          - #       445                      - - CCA GTA GGG CAA ACC TGC TGC CCA AGC CTC AA - #G GGA AGT TGG GCC TGC         1392                                                                       Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Ly - #s Gly Ser Trp Ala Cys               450              - #   455              - #   460                          - - TGC CAG CTG CCC CAT GCT GTG TGC TGT GAG GA - #C CGG CAG CAC TGT TGC         1440                                                                       Cys Gln Leu Pro His Ala Val Cys Cys Glu As - #p Arg Gln His Cys Cys           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - CCG GCC GGG TAC ACC TGC AAT GTG AAG GCG AG - #G ACC TGT GAG AAG        GAT     1488                                                                    Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Ar - #g Thr Cys Glu Lys Asp                          485  - #               490  - #               495              - - GTC GAT TTT ATC CAG CCT CCC GTG CTC CTG AC - #C CTC GGC CCT AAG GTT         1536                                                                       Val Asp Phe Ile Gln Pro Pro Val Leu Leu Th - #r Leu Gly Pro Lys Val                       500      - #           505      - #           510                  - - GGG AAT GTG GAG TGT GGA GAA GGG CAT TTC TG - #C CAT GAT AAC CAG ACC         1584                                                                       Gly Asn Val Glu Cys Gly Glu Gly His Phe Cy - #s His Asp Asn Gln Thr                   515          - #       520          - #       525                      - - TGT TGT AAA GAC AGT GCA GGA GTC TGG GCC TG - #C TGT CCC TAC CTA AAG         1632                                                                       Cys Cys Lys Asp Ser Ala Gly Val Trp Ala Cy - #s Cys Pro Tyr Leu Lys               530              - #   535              - #   540                          - - GGT GTC TGC TGT AGA GAT GGA CGT CAC TGT TG - #C CCC GGT GGC TTC CAC         1680                                                                       Gly Val Cys Cys Arg Asp Gly Arg His Cys Cy - #s Pro Gly Gly Phe His           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - TGT TCA GCC AGG GGA ACC AAG TGT TTG CGA AA - #G AAG ATT CCT CGC        TGG     1728                                                                    Cys Ser Ala Arg Gly Thr Lys Cys Leu Arg Ly - #s Lys Ile Pro Arg Trp                          565  - #               570  - #               575              - - GAC ATG TTT TTG AGG GAT CCG GTC CCA AGA CC - #G CTA CTG                  - #   1767                                                                    Asp Met Phe Leu Arg Asp Pro Val Pro Arg Pr - #o Leu Leu                                   580      - #           585                                         - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 589 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Trp Val Leu Met Ser Trp Leu Ala Phe Al - #a Ala Gly Leu Val Ala        1               5 - #                 10 - #                 15              - - Gly Thr Gln Cys Pro Asp Gly Gln Phe Cys Pr - #o Val Ala Cys Cys Leu                   20     - #             25     - #             30                  - - Asp Gln Gly Gly Ala Asn Tyr Ser Cys Cys As - #n Pro Leu Leu Asp Thr               35         - #         40         - #         45                      - - Trp Pro Arg Ile Thr Ser His His Leu Asp Gl - #y Ser Cys Gln Thr His           50             - #     55             - #     60                          - - Gly His Cys Pro Ala Gly Tyr Ser Cys Leu Le - #u Thr Val Ser Gly Thr       65                 - # 70                 - # 75                 - # 80       - - Ser Ser Cys Cys Pro Phe Ser Lys Gly Val Se - #r Cys Gly Asp Gly Tyr                       85 - #                 90 - #                 95              - - His Cys Cys Pro Gln Gly Phe His Cys Ser Al - #a Asp Gly Lys Ser Cys                  100      - #           105      - #           110                  - - Phe Gln Met Ser Asp Asn Pro Leu Gly Ala Va - #l Gln Cys Pro Gly Ser              115          - #       120          - #       125                      - - Gln Phe Glu Cys Pro Asp Ser Ala Thr Cys Cy - #s Ile Met Val Asp Gly          130              - #   135              - #   140                          - - Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Se - #r Cys Cys Glu Asp Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val His Cys Cys Pro His Gly Ala Ser Cys As - #p Leu Val His Thr        Arg                                                                                             165  - #               170  - #               175             - - Cys Val Ser Pro Thr Gly Thr His Thr Leu Le - #u Lys Lys Phe Pro Ala                  180      - #           185      - #           190                  - - Gln Lys Thr Asn Arg Ala Val Ser Leu Pro Ph - #e Ser Val Val Cys Pro              195          - #       200          - #       205                      - - Asp Ala Lys Thr Gln Cys Pro Asp Asp Ser Th - #r Cys Cys Glu Leu Pro          210              - #   215              - #   220                          - - Thr Gly Lys Tyr Gly Cys Cys Pro Met Pro As - #n Ala Ile Cys Cys Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp His Leu His Cys Cys Pro Gln Asp Thr Va - #l Cys Asp Leu Ile        Gln                                                                                             245  - #               250  - #               255             - - Ser Lys Cys Leu Ser Lys Asn Tyr Thr Thr As - #p Leu Leu Thr Lys Leu                  260      - #           265      - #           270                  - - Pro Gly Tyr Pro Val Lys Glu Val Lys Cys As - #p Met Glu Val Ser Cys              275          - #       280          - #       285                      - - Pro Glu Gly Tyr Thr Cys Cys Arg Leu Asn Th - #r Gly Ala Trp Gly Cys          290              - #   295              - #   300                          - - Cys Pro Phe Ala Lys Ala Val Cys Cys Glu As - #p His Ile His Cys Cys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Ala Gly Phe Gln Cys His Thr Glu Lys Gl - #y Thr Cys Glu Met        Gly                                                                                             325  - #               330  - #               335             - - Ile Leu Gln Val Pro Trp Met Lys Lys Val Il - #e Ala Pro Arg Arg Leu                  340      - #           345      - #           350                  - - Pro Asp Pro Gln Ile Leu Lys Ser Asp Thr Pr - #o Cys Asp Asp Phe Thr              355          - #       360          - #       365                      - - Arg Cys Pro Thr Asn Asn Thr Cys Cys Lys Le - #u Asn Ser Gly Asp Trp          370              - #   375              - #   380                          - - Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cy - #s Ser Asp Asn Gln His      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Cys Cys Pro Gln Gly Phe Thr Cys Leu Ala Gl - #n Gly Tyr Cys Gln        Lys                                                                                             405  - #               410  - #               415             - - Gly Asp Thr Met Val Ala Gly Leu Glu Lys Il - #e Pro Ala Arg Gln Thr                  420      - #           425      - #           430                  - - Thr Pro Leu Gln Ile Gly Asp Ile Gly Cys As - #p Gln His Thr Ser Cys              435          - #       440          - #       445                      - - Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Ly - #s Gly Ser Trp Ala Cys          450              - #   455              - #   460                          - - Cys Gln Leu Pro His Ala Val Cys Cys Glu As - #p Arg Gln His Cys Cys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Ar - #g Thr Cys Glu Lys        Asp                                                                                             485  - #               490  - #               495             - - Val Asp Phe Ile Gln Pro Pro Val Leu Leu Th - #r Leu Gly Pro Lys Val                  500      - #           505      - #           510                  - - Gly Asn Val Glu Cys Gly Glu Gly His Phe Cy - #s His Asp Asn Gln Thr              515          - #       520          - #       525                      - - Cys Cys Lys Asp Ser Ala Gly Val Trp Ala Cy - #s Cys Pro Tyr Leu Lys          530              - #   535              - #   540                          - - Gly Val Cys Cys Arg Asp Gly Arg His Cys Cy - #s Pro Gly Gly Phe His      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Cys Ser Ala Arg Gly Thr Lys Cys Leu Arg Ly - #s Lys Ile Pro Arg        Trp                                                                                             565  - #               570  - #               575             - - Asp Met Phe Leu Arg Asp Pro Val Pro Arg Pr - #o Leu Leu                              580      - #           585                                         - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 539 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bos tauru - #s                                                  (F) TISSUE TYPE: Kidney                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..537                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AAG TGG GGG TGT TGC CCG ATG CCC AAT GCC AT - #T TGC TGC TCC GAC CAC           48                                                                       Lys Trp Gly Cys Cys Pro Met Pro Asn Ala Il - #e Cys Cys Ser Asp His             1               5 - #                 10 - #                 15              - - CTG CAC TGC TGC CCC CAG AAC ACT GTG TGT GA - #C CTG ACC CAG AGT AAG           96                                                                       Leu His Cys Cys Pro Gln Asn Thr Val Cys As - #p Leu Thr Gln Ser Lys                        20     - #             25     - #             30                  - - TGC CTC TCC AAG GAG AAC GCT ACG GAC CTC CT - #C ACC AAG CTG CCC GCA          144                                                                       Cys Leu Ser Lys Glu Asn Ala Thr Asp Leu Le - #u Thr Lys Leu Pro Ala                    35         - #         40         - #         45                      - - CAC ACA GTG CAG GAT GTC AAG TGC GAC ATG GA - #G GTG AGC TGC CCA GAC          192                                                                       His Thr Val Gln Asp Val Lys Cys Asp Met Gl - #u Val Ser Cys Pro Asp                50             - #     55             - #     60                          - - GAC TAC ACC TGC TGC CGC CTA CAG TCC GGG GC - #C TGG GGC TGC TGC CCT          240                                                                       Asp Tyr Thr Cys Cys Arg Leu Gln Ser Gly Al - #a Trp Gly Cys Cys Pro            65                 - # 70                 - # 75                 - # 80       - - TTT GTG CAG GCC GTG TGC TGT GAG GAC CAT GT - #G CAC TGC TGC CCG TCC          288                                                                       Phe Val Gln Ala Val Cys Cys Glu Asp His Va - #l His Cys Cys Pro Ser                            85 - #                 90 - #                 95              - - GGG TTT AGG TGT GAC ACA GAG AAG GGT GTG TG - #T GAG CAG GGG ACC CGC          336                                                                       Gly Phe Arg Cys Asp Thr Glu Lys Gly Val Cy - #s Glu Gln Gly Thr Arg                       100      - #           105      - #           110                  - - CAG GTG CCG TGG ATG AAG AAA GCC CCA GCC CA - #C CTC AGC CTG CTG GAC          384                                                                       Gln Val Pro Trp Met Lys Lys Ala Pro Ala Hi - #s Leu Ser Leu Leu Asp                   115          - #       120          - #       125                      - - CTC GGA GCA GTG GAG GGG GAC GTC CCC TGT GA - #T AAC GTC ACC AGC TGT          432                                                                       Leu Gly Ala Val Glu Gly Asp Val Pro Cys As - #p Asn Val Thr Ser Cys               130              - #   135              - #   140                          - - CCT TCT TCC ACT ACC TGC TGT CGA CTC AAG TC - #T GGG GAG TGG GCC TGC          480                                                                       Pro Ser Ser Thr Thr Cys Cys Arg Leu Lys Se - #r Gly Glu Trp Ala Cys           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TGT CCT GCT CCA GAG GCT GTC TGC TGC TCG GA - #C CAC CAG CAC TGC        TGT      528                                                                    Cys Pro Ala Pro Glu Ala Val Cys Cys Ser As - #p His Gln His Cys Cys                          165  - #               170  - #               175              - - CCC CAA GAT AC             - #                  - #                      - #      539                                                                  Pro Gln Asp                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 179 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Lys Trp Gly Cys Cys Pro Met Pro Asn Ala Il - #e Cys Cys Ser Asp His        1               5 - #                 10 - #                 15              - - Leu His Cys Cys Pro Gln Asn Thr Val Cys As - #p Leu Thr Gln Ser Lys                   20     - #             25     - #             30                  - - Cys Leu Ser Lys Glu Asn Ala Thr Asp Leu Le - #u Thr Lys Leu Pro Ala               35         - #         40         - #         45                      - - His Thr Val Gln Asp Val Lys Cys Asp Met Gl - #u Val Ser Cys Pro Asp           50             - #     55             - #     60                          - - Asp Tyr Thr Cys Cys Arg Leu Gln Ser Gly Al - #a Trp Gly Cys Cys Pro       65                 - # 70                 - # 75                 - # 80       - - Phe Val Gln Ala Val Cys Cys Glu Asp His Va - #l His Cys Cys Pro Ser                       85 - #                 90 - #                 95              - - Gly Phe Arg Cys Asp Thr Glu Lys Gly Val Cy - #s Glu Gln Gly Thr Arg                  100      - #           105      - #           110                  - - Gln Val Pro Trp Met Lys Lys Ala Pro Ala Hi - #s Leu Ser Leu Leu Asp              115          - #       120          - #       125                      - - Leu Gly Ala Val Glu Gly Asp Val Pro Cys As - #p Asn Val Thr Ser Cys          130              - #   135              - #   140                          - - Pro Ser Ser Thr Thr Cys Cys Arg Leu Lys Se - #r Gly Glu Trp Ala Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Pro Ala Pro Glu Ala Val Cys Cys Ser As - #p His Gln His Cys        Cys                                                                                             165  - #               170  - #               175             - - Pro Gln Asp                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 341 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Gallus do - #mesticus                                           (F) TISSUE TYPE: Kidney                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..339                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AAG TGG GGT TGT TGC CCC ATG CCG AGA GGC GT - #G TGC TGC CGG GAT GAG           48                                                                       Lys Trp Gly Cys Cys Pro Met Pro Arg Gly Va - #l Cys Cys Arg Asp Glu             1               5 - #                 10 - #                 15              - - GAG CAC TGC TGT CCC CAC TCC ACC AGC TGT GA - #T TTG GAG CGC GGG CGC           96                                                                       Glu His Cys Cys Pro His Ser Thr Ser Cys As - #p Leu Glu Arg Gly Arg                        20     - #             25     - #             30                  - - TGT GTG TCC CCT ACG GGG GAC GTC CCC ATG GC - #C ACC AAA TTC CCG GCC          144                                                                       Cys Val Ser Pro Thr Gly Asp Val Pro Met Al - #a Thr Lys Phe Pro Ala                    35         - #         40         - #         45                      - - TGG AAG AGA CCG CGC GGT GCT GCG GCA CAG CC - #C CGG CTC CGC GTC CCA          192                                                                       Trp Lys Arg Pro Arg Gly Ala Ala Ala Gln Pr - #o Arg Leu Arg Val Pro                50             - #     55             - #     60                          - - GCA GTG GTT GGT GAC GTG AAG TGT GAC GAT GA - #G ATG AGC TGT CCC GAC          240                                                                       Ala Val Val Gly Asp Val Lys Cys Asp Asp Gl - #u Met Ser Cys Pro Asp            65                 - # 70                 - # 75                 - # 80       - - GGG AAC ACG TGC TGC AGG CTG AGC TCC GGG CA - #G TGG GGG TGC TGC CCG          288                                                                       Gly Asn Thr Cys Cys Arg Leu Ser Ser Gly Gl - #n Trp Gly Cys Cys Pro                            85 - #                 90 - #                 95              - - CTG GAG CAG GCC GTG TGC TGC CCC GAC CAC AT - #C CAC TGC TGC CCC CAA          336                                                                       Leu Glu Gln Ala Val Cys Cys Pro Asp His Il - #e His Cys Cys Pro Gln                       100      - #           105      - #           110                  - - GAT AC                - #                  - #                  - #               341                                                                  Asp                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Lys Trp Gly Cys Cys Pro Met Pro Arg Gly Va - #l Cys Cys Arg Asp Glu        1               5 - #                 10 - #                 15              - - Glu His Cys Cys Pro His Ser Thr Ser Cys As - #p Leu Glu Arg Gly Arg                   20     - #             25     - #             30                  - - Cys Val Ser Pro Thr Gly Asp Val Pro Met Al - #a Thr Lys Phe Pro Ala               35         - #         40         - #         45                      - - Trp Lys Arg Pro Arg Gly Ala Ala Ala Gln Pr - #o Arg Leu Arg Val Pro           50             - #     55             - #     60                          - - Ala Val Val Gly Asp Val Lys Cys Asp Asp Gl - #u Met Ser Cys Pro Asp       65                 - # 70                 - # 75                 - # 80       - - Gly Asn Thr Cys Cys Arg Leu Ser Ser Gly Gl - #n Trp Gly Cys Cys Pro                       85 - #                 90 - #                 95              - - Leu Glu Gln Ala Val Cys Cys Pro Asp His Il - #e His Cys Cys Pro Gln                  100      - #           105      - #           110                  - - Asp                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Cercopithecu - #s aethiops                                      (H) CELL LINE: BSC-1                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 28..108                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AGCTTCTGCA GGGGCGGGGC CTCCCCC ATG CCG CCC TCC GGG - #CTG CGG CTG             51                                                                                          - #            Met Pro Pro Se - #r Gly Leu Arg Leu                            - #              1    - #           5                        - - CTG CCG CTG CTG CTA CCG CTG CTG TGG CTA CT - #G GTG CTG ACG CCT AGC           99                                                                       Leu Pro Leu Leu Leu Pro Leu Leu Trp Leu Le - #u Val Leu Thr Pro Ser                10             - #     15             - #     20                          - - CGG CCG GCC GC             - #                  - #                      - #      110                                                                  Arg Pro Ala                                                                    25                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Le - #u Leu Leu Pro Leu Leu        1               5 - #                 10 - #                 15              - - Trp Leu Leu Val Leu Thr Pro Ser Arg Pro Al - #a                                       20     - #             25                                       __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a human epithelin 1 as depicted in SEQ ID NO: 4 from amino acid residue 282 to amino acid residue
 337. 2. The nucleic acid molecule of claim 1 wherein the nucleotide sequence is as depicted in SEQ ID NO: 3 from nucleotide residue numbers 844 to
 1011. 3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a human epithelin 2 as depicted in SEQ ID NO: 4 from amino acid residue 206 to amino acid residue
 262. 4. The nucleic acid molecule of claim 3 wherein the nucleotide sequence is as depicted in SEQ ID NO: 3 from nucleotide residue numbers 616 to
 786. 5. An isolated nucleic acid molecule encoding a human epithelin precursor having the amino acid sequence as depicted in SEQ ID NO. 4 from amino acid residue 1 to amino acid residue
 593. 6. The nucleic acid molecule encoding a human epithelin precursor of claim 5 comprising the nucleotide sequence as depicted in SEQ ID NO. 3 from nucleotide residue numbers 1 to
 1779. 7. An isolated nucleic acid molecule encoding a rat epithelin precursor having the amino acid sequence as depicted in SEQ ID NO. 2 from amino acid residue 1 to amino acid residue
 589. 8. The nucleic acid molecule encoding a rat epithelin precursor of claim 7 comprising the nucleotide sequence as depicted in SEQ ID NO. 1 from nucleotide residue numbers 1 to
 1767. 9. An isolated nucleic acid molecule encoding a mouse epithelin precursor having the amino acid sequence as depicted in SEQ ID NO. 6 from amino acid residue 1 to amino acid residue
 589. 10. The nucleic acid molecule encoding a mouse epithelin precursor of claim 9 comprising the nucleotide sequence as depicted in SEQ ID NO. 5 from nucleotide residue numbers 1 to
 1767. 